Polypeptides from mycrobacterium paratuberculosis

ABSTRACT

The invention relates to a polypeptide containing in its polypeptidic chain: the amino acid sequence of 101 amino acids of FIG.  8,  or a fragment of this sequence, this fragment being such that it is liable to be recognized by antibodies also recognizing the abovesaid sequence of 101 amino acids, but it is not recognized by antibodies respectively raised against  M. bovis, M. avium, M. phlei  and  M. tuberculosis,  and possibly against  M. leprae, M. intracellulare, M. scrofulaceum, M. fortuitum, M. gordonae  and  M. smegmatis;  it is liable to generate antibodies which also recognize the abovesaid sequence of 101 amino acids but which do not recognize  M. bovis, M. avium, M. Phlei  and  M. smegmatis;  it reacts with the majority of sera from cattle suffering from Johne&#39;s disease; or the polypeptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in so far as this modification does not alter the above-mentioned properties.

The invention relates to polypeptides and peptides, particularlyrecombinant ones, which can be used for the diagnosis ofparatuberculosis in cattle and possibly of Crohn's disease in humanbeings. The invention also relates to a process for preparing theabove-said polypeptides and peptides, which are in a state of biologicalpurity such that they can be used as part of the active principle in thepreparation of vaccines against paratuberculosis.

It also relates to nucleic acids coding for said polypeptides andpeptides.

Furthermore, the invention relates to the in vitro diagnostic methodsand kits using the above-said polypeptides and peptides and to thevaccines containing the above-said polypeptides and peptides as activeprinciple against paratuberculosis.

By “recombinant polypeptides or peptides” it is to be understood that itrelates to any molecule having a polypeptidic chain liable to beproduced by genetic engineering, through transcription and translation,of a corresponding DNA sequence under the control of appropriateregulation elements within an efficient cellular host. Consequently, theexpression “recombinant polypeptides” such as is used herein does notexclude the possibility for the polypeptides to comprise other groups,such as glycosylated groups.

The term “recombinant” indeed involves the fact that the polypeptide hasbeen produced by genetic engineering, particularly because it resultsfrom the expression in a cellular host of the corresponding nucleic acidsequences which have previously been introduced into an expressionvector used in said host.

Nevertheless, it must be understood that the polypeptides or thepeptides of the invention can be produced by a different process, forinstance by classical chemical synthesis according to methods used inthe protein synthesis or by proteolytic cleavage of larger molecules.

The expression “biologically pure” or “biological purity” means on theone hand a grade of purity such that the polypeptides can be used forthe production of vaccinating compositions and on the other hand theabsence of contaminants, more particularly of natural contaminants.

Paratuberculosis (Johne's disease) has been described as one of the mostserious diseases affecting the world cattle industry. Thismycobacteriosis produced by M. paratuberculosis is characterized by anileocoecal enteritis leading successively to emaciation, dysentery,cachexy and death (Chiodini R. J. et al., 1984, “Ruminantparatuberculosis (Johne's disease): the current status and futureprospects”, Cronell Vet. 74:218-262). Histological examination showsoedema, infiltration and thickening of the ileal mucosa, and hypertrophyand necrosis of intestinal lymphnodes. A miliary syndrome with diffusedparenchima granuloma in liver, spleen and lungs is not infrequent. Thehigh contagiousness of this disease is due to excretion of large numbersof bacteria from the intestinal tract: contaminated pastures propagatethe infection, rapidly producing live-stocks wherein infected animalsrepresent a large part of the population. Chronical dysentery is anadvanced stage of the disease, for epidemiological data suggest that thesubclinical cases, with little sign of intestinal alteration correspondto the majority of infected animals and frequently to a large proportionof a live-stock population.

Diagnosis of paratuberculosis is essential, especially in the absence ofclinical symptoms: it leads to identification of hidden bacterialshedders and avoids propagation of infection. Unfortunately, diagnosticindicators for early stages of the disease are missing. In fact,identification of the etiological agent (a slow grower) is a lengthyprocess, and histological examination of biopsy material is difficultand expensive. More interesting appear to be the immunologicalprocedures for analysis of humoral immune reactions (Brugère-Picoux J.,1987, “Le diagnostic de la paratuberculose chez les ruminants”, Rec.Méd. Vét. 163:539-546 Colgrave J. S. et al., 1989, “Paratuberculosis incattle: a comparison of three serologic tests with results of fecalculture”, Veterinary Microbiology 19:183-187). Although complementfixation and hemagglutination tests apparently lack both sensitivity andspecificity, immunoenzymometric methods for evaluation ofantimycobacterial antibodies seem to be more promising (Abbas B. et al.,1983, “Isolation of specific peptides from Mycobacteriumparatuberculosis protoplasm and their use in an enzyme linkedimmunosorbent assay for the detection of paratuberculosis (Johne'sdisease) in cattle”, Am. J. Vet. Res. 44:2229-2236; Colgrave J. S. etal., 1989, “Paratuberculosis in cattle: a comparison of three serologictests with results of fecal culture” Veterinary Microbiology,19:183-187; Yokomizo Y. et al., 1983, “Enzyme-linked immunosorbent assayfor detection of bovine immunoglobulin Gl antibody to a protoplasmicantigen of Mycobacterium paratuberculosis” Am. J. Vet. Res.44:2205-2207; Yokomizo Y. et al., 1985, “A method for avoidingfalse-positive reactions in an enzyme-linked immunosorbent assay (ELISA)for the diagnosis of bovine paratuberculosis” Japan, J. Vet. Sci.47:111-119).

Moreover, since slaughtering of cattle affected by tuberculosis (causedby M. bovis and/or M. tuberculosis), but not of those withparatuberculosis, is compulsory in Occidental countries, a distinctionat the immunological level between the two mycobacterial diseases isessential. Moreover, M. paratuberculosis is known to be geneticallyclose-related to M. avium (Chiodini R. J. et al., 1989, “The geneticrelationship between Mycobacterium paratuberculosis and the M. aviumcomplex” Acta Leprol. 7:249-251; Hurley S. S. et al., 1988,“Deoxyribonucleic acid-relatedness of Mycobacterium paratuberculosis toothers members of the family Mycobacteriaceae” Int. J. Syst. Bacteriol.38:143-146), which is a possible host of the intestinal tract ofruminants.

Taking into account the cross reactivity between M. paratuberculosis andmany other mycobacteria, it was a priori a difficult approach to find anantigen containing specific epitopes liable to be used as reagents forthe diagnosis of paratuberculosis, said reagents having no crossreactivity with other close related mycobacteria.

In addition to the above-mentioned aspects relative to paratuberculosisin cattle, M. paratuberculosis has been found to play an etiologic rolein at least some cases of Crohn's disease in human.

The disease originally described by Crohn and coworkers was a chronicalileitis producing hyperplastic granulomata of the intestine andlymphnodes. The syndrome presently known as Crohn's disease entailsinflammatory alterations of different organs of the digestive tract(month, larynx, esophagus, stomach, ileum and colon). Segments of themotive apparatus (joints, muscles and bones) can also be involved.Isolation of mycobacteria from patients affected by the Crohn's diseasehas been repeatedly related: in several instances isolates wereidentified as M. paratuberculosis. The induction by these isolates of asyndrome mimicking Crohn's disease in laboratory animals and primateshas been successful. In a recent review article (Chiodini R. J., 1989,“Crohn's disease and the mycobacterioses: a review and comparison of twodisease entities”, Clin. Microbiol. Rev. 2:90-117), Chiodini suggeststhis syndrome to be the expression of several pathological entities andconcludes, that, if Crohn's disease has a mycobacterial etiology, themost likely agent would be M. paratuberculosis.

At this present time, larger epidemiological investigation with an ELISAbased on a specific protein of M. paratuberculosis is expected to helpto solve the problem of the etiology of this enteritis resembling inmany respects the Johne's disease of cattle.

The expression “cattle” means ruminants, such as bovines, sheeps, goats,cervidae, but also include some non ruminant animals which may also beinfected by Johne's disease such as monkeys and horses.

An aspect of the invention is to provide recombinant polypeptides whichcan be used as purified antigens for the detection and control ofparatuberculosis.

Another aspect of the invention is to provide nucleic acids coding forthe peptidic chains of biologically pure recombinant polypeptides whichenable their preparation on a large scale.

Another aspect of the invention is to provide antigens which can be usedin serological tests as an in vitro rapid diagnosis of paratuberculosis,as well as in skin tests for in vivo diagnosis of paratuberculosis andas an immunogenic principle in vaccines.

Another aspect of the invention is to provide a rapid in vitrodiagnostic means for paratuberculosis, enabling it to discriminatebetween cattle suffering from tuberculosis from the ones suffering fromparatuberculosis.

Another aspect of the invention is to provide a rapid in vitrodiagnostic means for paratuberculosis, enabling it to discriminatebetween cattle suffering from paratuberculosis from the ones infected orcolonized by M. avium, M. bovis or M. tuberculosis or M. phlei.

Another aspect of the invention is to provide in vitro diagnostic meansfor patients suffering from Crohn's disease.

The invention relates to an antigen complex from M. paratuberculosis,named hereafter “the antigen A36”, liable to be obtained as follows:

sonication of bacterial suspensions of M. paratuberculosis to obtain ahomogenate (also named sonicate),

centrifugation of the above-mentioned homogenate to obtain a supernatant(which corresponds to the cytoplasm of the bacteria),

RNAase digestion of the above-mentioned supernatant,

fractionation of the digested supernatant, for instance by gel exclusionchromatography, for instance on Sepharose 6B columns,

recovery of the antigen complex (A36) which is the excluded fraction ofthe fractionation.

It is to be noted that the antigen complex hereabove defined correspondsto the TMA complex (thermostable macromolecular antigens), belonging toa family of complexes present in all mycobacteria and consisting of orcontaining lipid, polysaccharide and protein moieties.

The proteic part of the antigen complex of the invention can befractionated and visualized as follows:

fractionation of the proteins of the above-mentioned antigen complex byelectrophoresis in a gel, for instance 10% polyacrylamide gels to obtainthe protein on bands,

detection of the proteins by staining for instance with Coomassie blue.

The polypeptides of the invention contain in their polypeptidic chain:

the amino acid sequence of 101 amino acids of FIG. 8,

or a fragment of this sequence, this fragment being such that:

it is liable to be recognized by antibodies also recognizing theabovesaid sequence of 101 amino acids, but it is not recognized byantibodies raised respectively against M. bovis, M. avium, M. phlei andM. tuberculosis,

it is liable to generate antibodies which also recognizing the abovesaidsequence of 101 amino acids but which do not recognize M. bovis, M.avium, M. phlei and M. tuberculosis,

it reacts with the majority of sera from cattle suffering from Johne'sdisease,

or the polypeptidic sequences resulting from the modification bysubstitution and/or by addition and/or by deletion of one or severalamino acids in so far as this modification does not alter theabove-mentioned properties.

Recognition of one of the above-mentioned fragments by theabove-mentioned antibodies—or of the abovesaid sequence of 101 aminoacids by the above-mentioned antibodies—means that the above-mentionedfragment can form a complex with one of the above-said antibodies.

The formation of the complex antigen (i.e. the sequence of 101 aminoacids (SEQ ID NO:5) or of the above-said fragment)—antibody and thedetection of the existence of a formed complex can be done according toclassical techniques such as the ones using a marker labeled byradioactive isotopes or by an enzyme.

Hereafter is also given in a non limitative way, a test for givingevidence of the fact that polypeptides of the invention are recognizedselectively by the majority of the sera from cattle suffering fromJohne's disease (immunodominant polypeptides), for instance bovines.

This test is an immunoblotting (Western blotting) analysis, in the casewhere the polypeptides of the invention are obtained by recombinanttechniques. This test can also be used for polypeptides of the inventionobtained by a different preparation process. After sodium dodecylsulfate-polyacrylamide gel electrophoresis, polypeptides of theinvention are blotted onto nitrocellulose membranes (Hybond C.(Amersham)) as described by Towbin H. et al., 1979, “Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications”, Proc. Natl. Acad. Sci. USA76:4350-4354. The expression of polypeptides of the invention fused toβ-galactosidase in E. coli Y1089, is visualized by the binding of apolyclonal rabbit anti-A36 antiserum (or polyclonal rabbitanti-homogenate antiserum defined hereafter in the examples, orpolyclonal rabbit anti-βgal-p362 antiserum, defined hereafter in theexamples) (1:1,000) or by using a monoclonal anti-β-galactosidaseantibody (Promega). The secondary antibody (anti-rabbit immunoglobulin Gand anti-mouse immunoglobulin G respectively, both alkaline phosphataseconjugated) is diluted as recommended by the supplier (Promega). Colourreaction is developed by adding NBT/BCIP (Nitro Blue Tetrazolium 5-bromo4-chloro-3-indolyl phosphate [Promega]) using conditions recommended bysuppliers.

In order to identify selective recognition of polypeptides of theinvention and of fusion proteins of the invention by sera of bovinesuffering from Johne's disease, nitrocellulose sheets are incubatedovernight with each of these sera (1:50) (after blocking a specificprotein-binding sites).

Reactive areas on the nitrocellulose sheets are revealed by incubationwith peroxidase conjugated goat anti-bovine immunoglobulin G antibody(Dakopatts, Copenhagen, Denmark) (1:200) for 4 h, and after repeatedwashings, color reaction is developed by adding α-chloronaphtol (Bio-RadLaboratories, Richmond, Calif.) in the presence of hydrogen peroxide.

The non-recognition of the antibodies raised against the above-mentionedfragments of the invention by M. bovis, M. avium, M. phlei and M.tuberculosis and by other mycobacteria can be done according to aprocess detailed in the examples.

As to the non-recognition of the above-mentioned fragments of theinvention by antibodies raised respectively against M. bovis, M. avium,M. phlei and M. tuberculosis or other mycobacteria, it can also be doneaccording to a process detailed in the examples.

Advantageous above-defined fragments of the invention are liable not tobe recognized by antibodies raised against other mycobacteria such as M.leprae, M. intracellulare, M. scrofulaceum, M. fortuitum, M. gordonaeand M. smegmatis, and are liable to generate antibodies which do notrecognize M. leprae, M. intracellulare, M. scrofulaceum, M. fortuitum,gordonae and M. smegmatis.

It goes without saying that the free reactive functions which arepresent in some of the amino acids, which are part of the constitutionof the polypeptides of the invention, particularly the free carboxylgroups which are carried by the groups Glu and Asp or by the C-terminalamino acid on the one hand and/or the free NH₂ groups carried by theN-terminal amino acid or by amino acids inside the peptidic chain, forinstance Lys, on the other hand, can be modified in so far as thismodification does not alter the above mentioned properties of thepolypeptide.

The molecules which are thus modified are naturally part of theinvention. The above mentioned carboxyl groups can be acylated oresterified.

Other modifications are also part of the invention. Particularly, theamine or carboxyl functions or both of terminal amino acids can bethemselves involved in the bond with other amino acids. For instance,the N-terminal amino acid can be linked to the C-terminal amino acid ofanother peptide comprising from 1 to several amino acids.

Furthermore, any peptidic sequences resulting from the modification bysubstitution and/or by addition and/or by deletion of one or severalamino acids of the polypeptides according to the invention are part ofthe invention in so far as this modification does not alter the abovementioned properties of said polypeptides.

The polypeptides according to the invention can be glycosylated or not,particularly in some of their glycosylation sites of the type Asn-X-Seror Asn-X-Thr, X representing any amino acid.

An advantageous recombinant polypeptide of the invention is constitutedby the sequence represented on FIG. 8, extending from the extremityconstituted by amino acid at position (1) to the extremity constitutedby amino acid at position (101), or by the following peptides:

Glu-Phe-Pro-Gly-Gly-Gln-Gln-His-Ser-Pro-Gln, (position 1 to 11 on FIG.8) (SEQ ID NO:12)

Gln-Gln-Ser-Tyr-Gly-Gln-Glu-Pro-Ser-Ser-Pro-Ser-Gly-Pro-Thr-Pro-Ala(position 85 to 101 on FIG. 8) (SEQ ID NO:13).

It is to be noted that this polypeptide is derived from the expressionproduct of a DNA derived from the nucleotide sequence coding for apolypeptide of 10 kDa being the carboxy terminal part of a 34 kDaprotein of M. paratuberculosis, defined hereafter.

An advantageous recombinant polypeptide of the invention ischaracterized by the fact that:

it contains the amino sequence of 101 amino acids of FIG. 8 (SEQ ID NO:5) as its C-terminal part,

it has a molecular weight of about 34 kDa, in SDS-PAGE,

it is coded by a nucleotide sequence liable to hybridize with thecomplementary strand of the sequence of FIG. 11 (SEQ ID NO:2),

it reacts with the majority of sera from cattle suffering from Johne'sdisease,

it is advantageously liable to elicit a cellular immune response insensitized subjects.

Subjects can be either test animals such as mice or guinea pigs orcattle or human beings.

“Sensitized” means that these subjects have been in contact previouslywith M. paratuberculosis, resulting in a priming of the cellular immunesystem.

Sensitization can be induced by inoculating the subjects with killed orattenuated M. paratuberculosis or it can result from a natural infectionwith M. paratuberculosis.

A positive cellular immune response to the polypeptides of the inventioncan be detected for example in vivo by a delayed—type hypersensitivityreaction upon skintesting with the polypeptides of the invention or invitro by proliferation of peripheral blood lymphocytes isolated fromsensitized subjects, in response to the added polypeptides.

An advantageous recombinant polypeptide of the invention contains or isconstituted by the amino acid sequence of FIG. 11 (SEQ ID NO:11).

Another advantageous recombinant polypeptide of the invention containsor is constituted by the amino acid sequence extending from amino acidat position (1) to the amino acid at position (199), of FIG. 11 (SEQ IDNO:11).

It is to be noted that this polypeptide is a 34 kDa protein which ispresent in the proteic part of the TMA complex of M. paratuberculosis(A36).

Hereafter is given, in a non limitative way, a process for preparingthis 34 kDa protein of the invention.

The DNA sequence (306 bp) coding for p362 (SEQ ID NO:4), being thecarboxyterminal end of the 34 kDa protein has been determined (SEQ IDNO:5) (see FIG. 8). It contains a unique ApaI (GGGCCC) site at position141.

Using this information, the full gene coding for the 34 kDa protein canbe isolated as follows:

An oligonucleotide coding for a stretch of at least 30 bp, situatedwithin the region EcoRI-ApaI (1-141 bp) of the known sequence, issynthesized.

It is labeled and used as a probe to hybridize to the DNA of M.paratuberculosis (strain ATCC 19698), which has previously been cut byApaI, separated by agarose gel electrophoresis, denatured andtransferred to a nylon membrane.

This hybridization indicates a band on the nylon membrane of around 1500bp, which contains the coding part for the rest of the 34 kDa protein.After having located this 1500 bp fragment, flanked by 2 ApaI sites, inthe agarose gel, it is isolated from the gel, purified and subcloned inthe ApaI site of the sequencing vector pBluescript SK⁺.

After sequencing of this fragment, the coding region, starting with theinitiation codon ATG or GTG, is delineated. Using a restriction sitenear the initiation codon (5′ end), naturally present or created bysite-directed mutagenesis, and the ApaI site at the 3′ end, the DNAfragment coding for the N-terminal part of the protein (about 750 bp) isexcised from pBluescript SK⁺, and purified. It is ligated to the ApaIsite of the fragment coding for the C-terminal part of p362 (142-306,FIG. 8), that for example has been prepared synthetically.

The complete gene coding for the 34 kDa protein (about 910 bp) issubcloned in an expression vector and expressed in E. coli. Therecombinant 34 kDa protein is then purified.

The invention also relates to the amino acid sequences constituted bythe above mentioned polypeptides and a protein or an heterologoussequence with respect to said polypeptide, said protein or heterologoussequence comprising for instance from about 1 to about 1100 amino acids.These amino acid sequences will be called fusion proteins.

In an advantageous fusion protein of the invention, the heterologousprotein is β-galactosidase.

The invention also relates to a nucleic acid characterized by the factthat it comprises or is constituted by:

a nucleotide chain liable to hybridize with the nucleotide chain codingfor the polypeptides according to the invention, or

a nucleotide chain coding for the polypeptides according to theinvention, or

the complementary sequences of the above nucleotide chains.

The invention also relates to nucleic acids comprising nucleotidesequences which hybridize with the nucleotide sequences coding for anyof the above mentioned polypeptides under the following hybridizationconditions:

hybridization and wash medium:

a preferred hybridization medium contains about 3×SSC [SSC=0.15 M sodiumchloride, 0.015 M sodium citrate, pH 7], about 25 mM of phosphate bufferpH 7.1, and 20% deionized formamide, 0.02% Ficoll, 0.02% BSA, 0.02%polyvinylpyrrolidone and about 0.1 mg/ml sheared denatured salmon spermDNA,

a preferred wash medium contains about 3×SSC, about 25 mM phosphatebuffer, pH 7.1 and 20% deionized formamide;

hybridization temperature (HT) and wash temperature (WT) for the nucleicacids of the invention defined by x-y: i.e. by the sequence extendingfrom the extremity constituted by the nucleotide at position (x) to theextremity constituted by the nucleotide at position (y) represented onFIGS. 7A (SEQ ID NO:1), 7B (SEQ ID NO:2) or 7C (SEQ ID NO:3):

1-306 (for FIGS. 7B (SEQ ID NO:16) and 7c (SEQ ID NO:17)) or

HT=WT=65° C.

1-307 (for FIG. 7A (SEQ ID NO:14))

1-507 (for FIGS. 7B (SEQ ID NO:2) and 7c (SEQ ID NO:3))

HT=WT=65° C.

1-508 (for FIG. 7A (SEQ ID NO:1))

The above mentioned temperatures are to be considered as approximately±5° C.

It is to be noted that in the above defined nucleic acids, as well as inthe hereafter defined nucleic acids, the nucleotide sequences which arebrought into play are such that T can be replaced by U.

A group of preferred nucleic acids of the invention comprises one atleast of the following nucleotide sequences:

the one extending from the extremity constituted by nucleotide atposition (1) to the extremity constituted by nucleotide at position(307) (SEQ ID NO:14) represented in FIG. 7A,

the one extending from the extremity constituted by nucleotide atposition (1) to the extremity constituted by nucleotide at position(508) (SEQ ID NO:1) represented in FIG. 7A, wherein

X and E represent phosphodiester bonds,

Y and F represent respectively G and C,

Z and H represent respectively C and G, or

X and E represent respectively G and C,

Y and F represent respectively C and G,

Z and H represent phosphodiester bonds.

A group of preferred nucleic acids of the invention comprises one atleast of the following nucleotide sequences:

the one extending from the extremity constituted by nucleotide atposition (1) to the extremity constituted by nucleotide at position(306) (SEQ ID NO:15) represented in FIG. 7B,

the one extending from the extremity constituted by nucleotide atposition (1) to the extremity constituted by nucleotide at position(507) represented in FIG. 7B (SEQ ID NO:2).

The nucleotide sequence represented in FIG. 7B corresponds to the onerepresented in FIG. 7A, wherein

X and E represent phosphodiester bonds,

Y and F represent respectively G and C,

Z and H represent respectively C and G.

The invention also relates to a nucleic acid characterized by the factthat it comprises or is constituted by a nucleotide chain,

extending from the extremity constituted by nucleotide at position (1)to the extremity constituted by nucleotide at position (306) (SEQ IDNO:16) on FIG. 7C, or

extending from the extremity constituted by nucleotide at position (1)to the extremity constituted by nucleotide at position (507) (SEQ IDNO:3) on FIG. 7C.

The nucleotide sequence represented on FIG. 7C corresponds to the onerepresented on FIG. 7A, wherein

X and E represent respectively G and C,

Y and F represent respectively C and G,

Z and H represent phosphodiester bonds.

The invention also relates to a nucleic acid which comprises or isconstituted by:

a nucleotide sequence liable to hybridize with the complementary strandof the nucleotide sequence of FIG. 11 (SEQ ID NO:10), or with thecomplementary strand of the nucleotide sequence extending fromnucleotide at position (742) to nucleotide at position (1338) (SEQ IDNO:17) of FIG. 11,

the nucleotide sequence of FIG. 11 or the nucleotide sequence extendingfrom nucleotide at position (742) to nucleotide at position (1338) (SEQID NO:17) of FIG. 11,

the complementary sequences of the nucleotide sequences above-defined.

From the nucleic acids of the invention, probes (i.e. cloned orsynthetic oligonucleotides) can be inferred.

These probes can be from 15 to the maximum number of nucleotides of theselected nucleic acids. The oligonucleotides can also be used either asamplification primers in the PCR technique (PCR, Mullis and Faloona,Methods in Enzymology, vol. 155, p. 335, 1987) to generate specificenzymatically amplified fragments and/or as probes to detect fragmentsamplified between bracketing oligonucleotide primers.

The specificity of a PCR-assisted hybridization assay can be controlledat different levels.

The amplification process or the detection process or both can bespecific. The latter case giving the higher specificity is preferred.

The invention also relates to any recombinant nucleic acid containing atleast one of the nucleic acids of the invention combined to or insertedin a heterologous nucleic acid.

The invention relates more particularly to recombinant nucleic acid suchas defined, in which the nucleotide sequence of the invention ispreceded by a promoter (particularly an inducible promoter) under thecontrol of which the transcription of said sequence is liable to beprocessed and possibly followed by a sequence coding for transcriptiontermination signals.

The invention also relates to the recombinant nucleic acids in which thenucleic acid sequences coding for the polypeptide of the invention andpossibly the signal peptide, are recombined with control elements whichare heterologous with respect to the ones to which they are normallyassociated with in the mycobacterial genome and, more particularly, theregulation elements adapted to control their expression in the cellularhost which has been chosen for their production.

The invention also relates to recombinant vectors, particularly forcloning and/or expression, comprising a vector sequence, notably of thetype plasmid, cosmid or phage or virus DNA, and a recombinant nucleicacid of the invention, inserted in one of the non essential sites forits replication.

According to an advantageous embodiment of the invention, therecombinant vector contains necessary elements to promote the expressionin a cellular host of polypeptides coded by nucleic acids according tothe invention inserted in said vector and notably a promoter recognizedby the RNA polymerase of the cellular host, particularly an induciblepromoter and possibly a sequence coding for transcription terminationsignals and possibly a signal sequence and/or an anchoring sequence.

According to another additional embodiment of the invention, therecombinant vector contains the elements enabling the expression by E.coli of a fusion protein consisting of the polypeptide ofβ-galactosidase or part thereof linked to a polypeptide coded by anucleic acid according to the invention.

The invention also relates to a cellular host, chosen from amongbacteria such as E. coli or chosen from among eukaryotic organism, suchas CHO cells or insect cells, which is transformed by a recombinantvector according to the invention, and containing the regulationelements enabling the expression of the nucleotide sequence coding forthe polypeptide according to the invention in this host.

The invention relates to an expression product of a nucleic acidexpressed by a transformed cellular host according to the invention.

The invention also relates to a process for preparing a recombinantpolypeptide according to the invention comprising the following steps:

the culture in an appropriate medium of a cellular host which haspreviously been transformed by an appropriate vector containing anucleic acid according to the invention,

the recovery of the polypeptide produced by the above said transformedcellular host from the above said culture medium, or from the cellularhost, and

possibly the purification of the polypeptide produced, eventually bymeans of immobilized metal ion affinity chromatography (IMAC).

The polypeptides of the invention can be prepared according to theclassical techniques in the field of peptide synthesis.

The synthesis can be carried out in homogeneous solution or in solidphase.

For instance, the synthesis technique in homogeneous solution which canbe used is the one described by Houbenweyl in the book titled “Methodeder organischen chemie” (Method of organic chemistry) edited by E.Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.

The polypeptides of the invention can also be prepared in solid phaseaccording to the method described by Atherton & Shepard in their booktitled “Solid phase peptide synthesis” (Ed. IRL Press, Oxford, NY,Tokyo, 1989).

The invention also relates to a process for preparing the nucleic acidsaccording to the invention.

A suitable method for chemically preparing the single-stranded nucleicacids (containing at most 100 nucleotides of the invention) comprisesthe following steps:

DNA synthesis using the automatic β-cyanoethyl phosphoramidite methoddescribed in Bioorganic Chemistry 4; 274-325, 1986.

In the case of single-stranded DNA, the material which is obtained atthe end of the DNA synthesis can be used as such.

A suitable method for chemically preparing the double-stranded nucleicacids (containing at most 100 bp of the invention) comprises thefollowing steps:

DNA synthesis of one sense oligonucleotide using the automaticβ-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4;274-325, 1986, and DNA synthesis of one anti-sense oligonucleotide usingsaid above-mentioned automatic β-cyanoethyl phosphoramidite method,

combining the sense and anti-sense oligonucleotides by hybridization inorder to form a DNA duplex,

cloning the DNA duplex obtained into a suitable plasmid vector andrecovery of the DNA according to classical methods, such as restrictionenzyme digestion and agarose gel electrophoresis.

A method for the chemical preparation of nucleic acids of length greaterthan 100 nucleotides—or bp, in the case of double-stranded nucleicacids—comprises the following steps:

assembling of chemically synthesized oligonucleotides, provided at theirends with different restriction sites, the sequences of which arecompatible with the succession of amino acids in the natural peptide,according to the principle described in Proc. Nat. Acad. Sci. USA 80;7461-7465, 1983,

cloning the DNA thereby obtained into a suitable plasmid vetor andrecovery of the desired nucleic acid according to classical methods,such as restriction enzyme digestion and agarose gel electrophoresis.

The invention also relates to antibodies themselves formed against thepolypeptides according to the invention, and characterized by the factthat they recognize neither M. bovis, nor M. avium, nor M. phlei, nor M.tuberculosis.

It goes without saying that this production is not limited to polyclonalantibodies.

It also relates to any monoclonal antibody produced by any hybridomaliable to be formed according to classical methods from splenic cells ofan animal, particularly of a mouse or rat, immunized against thepurified polypeptide of the invention on the one hand, and of cells of amyeloma cell line on the other hand, and to be selected by its abilityto produce the monoclonal antibodies recognizing the polypeptide whichhas been initially used for the immunization of the animals.

The invention also relates to any antibody of the invention labeled byan appropriate label of the enzymatic, fluorescent or radioactive type.

The polypeptide which is advantageously used to produce antibodies,particularly monoclonal antibodies, is the one or part of the oneextending from the extremity constituted by amino acid at position (1)to the extremity constituted by amino acid at position (101) (SEQ IDNO:5) represented on FIG. 8.

Variations of this peptide are also possible depending on its intendeduse. For example, if the peptide is to be used to raise antisera, thepeptide may be synthesized with an extra cysteine residue added. Thisextra cysteine residue is preferably added to the amino terminus andfacilitates the coupling of the peptide to a carrier protein which isnecessary to render the small peptide immunogenic. If the peptide is tobe labeled for use in radioimmune assays, it may be advantageous tosynthesize the protein with a tyrosine attached to either the amino orcarboxyl terminus to facilitate iodination. This peptide possessestherefore the primary sequence of the peptide above-mentioned but withadditional amino acids which do not appear in the primary sequence ofthe protein and whose sole function is to confer the desired chemicalproperties to the peptide.

The invention also relates to a process for detecting in vitroantibodies related to paratuberculosis in a biological sample of ananimal liable to contain them, this process comprising

contacting the biological sample with a polypeptide or a peptideaccording to the invention, or the expression product of the invention,under conditions enabling an in vitro immunological reaction betweensaid polypeptide and the antibodies which are possibly present in thebiological sample and

the in vitro detection of the antigen/antibody complex which may beformed.

Preferably, the biological medium is constituted by an animal serum, andparticularly by bovine serum.

The detection can be carried out according to any classical process.

By way of example a preferred method brings into play an immunoenzymaticprocess according to ELISA technique or immunofluorescent orradioimmunlogical (RIA) or the equivalent ones.

Thus the invention also relates to any polypeptide according to theinvention labeled by an appropriate label of the enzymatic, fluorescent,radioactive . . . type.

Such a method for detecting in vitro antibodies related toparatuberculosis comprises for instance the following steps:

deposit of determined amounts of a polypeptidic composition according tothe invention in the wells of a titration microplate,

introduction into said wells of increasing dilutions of the serum to bediagnosed,

incubation of the microplate,

repeated rinsing of the microplate,

introduction into the wells of the microplate of labeled antibodiesagainst the blood immunoglobulins,

the labeling of these antibodies being based on the activity of anenzyme which is selected from among the ones which are able to hydrolyzea substrate by modifying the absorption of the radiation of this latterat least at a given wave length,

detection by comparing with a control standard of the amount ofhydrolyzed substrate.

The invention also relates to a process for detecting and identifying invitro antigens of M. paratuberculosis in an animal biological sampleliable to contain them, this process comprising:

contacting the biological sample with an appropriate antibody of theinvention under conditions enabling an in vitro immunological reactionbetween said antibody and the antigens of M. paratuberculosis which arepossibly present in the biological sample and

the in vitro detection of the antigen/antibody complex which may beformed.

Preferably, the biological medium is constituted by serum or faeces,milk or urine, particularly of bovine origin.

Appropriate antibodies are advantageously monoclonal antibodies directedagainst the above-mentioned peptide.

The invention also relates to an additional method for the in vitrodiagnosis of paratuberculosis in an animal liable to be infected byMycobacterium paratuberculosis comprising:

contacting a biological sample taken from an animal with a polypeptideor a peptide of the invention, or the expression product of theinvention, under conditions enabling an in vitro immunological reactionbetween said polypeptide or peptide and the antibodies which arepossibly present in the biological sample and

the in vitro detection of the antigen/antibody complex which haspossibly been formed.

To carry out the in vitro diagnostic method for paratuberculosis in ananimal liable to be infected by Mycobacterium paratuberculosis, thefollowing necessary or kit can be used, said necessary or kitcomprising:

a polypeptide or a peptide according to the invention, or the expressionproduct of the invention,

reagents for making a medium appropriate for the immunological reactionto occur,

reagents enabling to detect the antigen/antibody complex which has beenproduced by the immunological reaction, said reagents possibly having alabel, or being liable to be recognized by a labeled reagent, moreparticularly in the case where the above mentioned polypeptide orpeptide is not labeled.

The invention also relates to an additional method for the in vitrodiagnosis of paratuberculosis in an animal liable to be infected by M.paratuberculosis, comprising the following steps:

contacting a biological sample of said animal with an appropriateantibody of the invention under conditions enabling an in vitroimmunological reaction between said antibody and the antigens of M.paratuberculosis which are possibly present in the biological sample and

the in vitro detection of the antigen/antibody complex which may beformed.

To carry out the in vitro diagnostic method for paratuberculosis in ananimal liable to be infected by Mycobacterium paratuberculosis, thefollowing necessary or kit can be used, said necessary or kitcomprising:

an antibody of the invention,

reagents for making a medium appropriate for the immunological reactionto occur,

reagents enabling to detect the antigen/antibody complexes which havebeen produced by the immunological reaction, said reagent possiblyhaving a label or being liable to be recognized by a labeled reagent,more particularly in the case where the above-mentioned antibody is notlabeled.

An advantageous kit for the in vitro diagnosis of paratuberculosiscomprises:

at least a suitable solid phase system, e.g. a microtiter-plate fordeposition thereon of the biological sample to be diagnosed in vitro,

a preparation containing one of the monoclonal antibodies of theinvention,

a specific detection system for said monoclonal antibody,

appropriate buffer solutions for carrying out the immunological reactionbetween a test sample and said monoclonal antibody on the one hand, andthe bonded monoclonal antibodies and the detection system on the otherhand.

The invention also relates to a kit, as described above, also containinga preparation of one of the polypeptides or peptides of the invention,said antigen of the invention being either a standard (for quantitativedetermination of the antigen of M. paratuberculosis which is sought) ora competitor, with respect to the antigen which is sought, for the kitto be used in a competition dosage process.

The invention also relates to a method for the in vitro diagnosis ofCrohn's disease in a patient liable to be infected by Mycobacteriumparatuberculosis comprising the following steps:

contacting the biological sample with an appropriate antibody accordingto the invention, under conditions enabling an in vitro immunologicalreaction between said antibody and the antigens of M. paratuberculosiswhich are possibly present in the biological sample and

the in vitro detection of the antigen/antibody complex which may beformed.

The invention also relates to a method for the in vitro diagnosis ofCrohn's disease in a patient liable to be infected by M.paratuberculosis, comprising the following steps:

contacting a biological sample taken from a patient with a polypeptideor peptide according to the invention, or the expression product of theinvention, under conditions enabling an in vitro immunological reactionbetween said polypeptide and the antibodies which are possibly presentin the biological sample and

the in vitro detection of the antigen/antibody complex which has beenpossibly formed.

The invention also relates to a necessary or kit for an in vitrodiagnosis method of Crohn's disease in a patient liable to be infectedby Mycobacterium paratuberculosis, said necessary or kit comprising:

an antibody according to the invention,

reagents for making a medium appropriate for the immunological reactionto occur,

reagents enabling to detect the antigen/antibody complexes which havebeen produced by the immunological reaction, said reagents possiblyhaving a label or being liable to be recognized by a labeled reagent,more particularly in the case where the above-mentioned antibody is notlabeled.

The invention also relates to a necessary or kit for an in vitrodiagnosis method of Crohn's disease in a patient liable to be infectedby Mycobacterium paratuberculosis said necessary or kit comprising:

a polypeptide or a peptide according to the invention, or the expressionproduct of the invention,

reagents for making a medium appropriate for the immunological reactionto occur,

reagents enabling to detect the antigen/antibody complex which has beenproduced by the immunological reaction, said reagents possibly having alabel, or being liable to be recognized by a labeled reagent, moreparticularly in the case where the above mentioned polypeptide is notlabeled.

The invention also relates to an immunogenic composition comprising apolypeptide or a peptide according to the invention, or the expressionproduct of the invention, in association with a pharmaceuticallyacceptable vehicle.

The invention also relates, to a vaccine composition comprising amongother immunogenic principles anyone of the polypeptides or peptides ofthe invention or the expression product of the invention, possiblycoupled to a natural protein or to a synthetic polypeptide having asufficient molecular weight so that the conjugate is able to induce invivo the production of antibodies neutralizing Mycobacteriumparatuberculosis, or induce in vivo a protective cellular immuneresponse by activating M. paratuberculosis antigen-responsive T cells.

The invention also relates to a necessary or kit for the diagnosis ofprior exposure of an animal to M. paratuberculosis, said necessary orkit containing a preparation of at least one of the polypeptides orpeptides of the invention, or the expression product of the invention,with said preparation being able to induce in vivo after beingintradermally injected to an animal a delayed type hypersensitivityreaction, at the site of injection, in case the animal has had priorexposure to M. paratuberculosis.

Other characteristics and advantages of the invention will appear in thefollowing examples and the figures illustrating the invention.

LEGENDS TO FIGURES

FIG. 1 (1) represents the two-dimensional cross immunoelectrophoresis(CIE) of total cytoplasm (the supernatant fraction obtained aftercentrifugation of the sonicate) from M. paratuberculosis and FIG. 1(2)represents the two-dimensional cross immunoelectrophoresis of theexclusion fraction obtained by gel exclusion chromatography of the samecytoplasm.

In the second dimension (upward in the figure), migration was made in agel containing rabbit antiserum directed against the mycobacterialsonicate. Preparations in 1 and 2 contained 10 μg of proteins. Thisfigure identifies the TMA complex of M. paratuberculosis (A36) presentin the exclusion fraction.

FIG. 2 represents the serological analysis of infected animals withpolypeptide p362. Multiwell plates were coated with 4 μg ofproteins/well of E. coli-a362 total cytoplasm (white) or E. coli-controltotal cytoplasm (black). Samples of diluted (1/400) bovine serapreviously exhausted by incubation with E. coli-control homogenate (saidhomogenate and total cytoplasm being obtained in the same way as M.paratuberculosis homogenate and total cytoplasm as described above) wereadded, followed by washing, incubation with labeled anti-bovine Ig,peroxidase reagents and spectrophotometric reading at 450 nm.

The following sera were used: asymptomatic non-excretory (sample 1),asymptomatic excretory (samples 2 to 13), symptomatic excretory (samples14 to 24) and healthy bovine (samples 26 to 32).

FIG. 3 represents the serological analysis of infected animals with aA36-based immunoassay.

Multiwell plates were coated with comparable amounts (0.5 μg totalproteins/well) of: M. paratuberculosis total cytoplasm (black), A36(white) and B. subtilis total cytoplasm (control: hatched). Samples ofdiluted (1/400) bovine sera previously exhausted by incubation with B.subtilis homogenate (said homogenate and total cytoplasm being obtainedin the same way as M. paratuberculosis homogenate and total cytoplasm asabove-described) were added, followed by washing, incubation withlabeled anti-bovine Ig, peroxidase reagents and spectrophotometricreading at 450 nm. The following bovine sera were used:

a) symptomatic-excretory forms of paratuberculosis (samples 1 to 7); b)asymptomatic-excretory forms (samples 8 to 12); and c) healthy cattle(samples 13 to 15). Mean values of absorbance and standard deviationsare the results of 4 repeats.

FIG. 4 represents the recognition of different A36 proteins by the seraof infected bovines. A36 proteins from M. paratuberculosis werefractionated by gel electrophoresis and transferred to nitrocellulose.Membranes were incubated with sera from uninfected (lane 8) or infectedanimals (lanes 4 to 7), either pre-absorbed (lane 7) or not (lanes 4, 5,6) with a mixture of homogenates of M. avium, M. bovis and M. phlei.Membrane-bound primary Ig were revealed by labeled secondary Ig. Sera ofinfected animals were as follows: asymptomatic-non excretory (lane 4),asymptomatic-excretory (lane 5), and symptomatic-excretory (lane 6, 7)cases of paratuberculosis. Reference molecular weight standards (lane 1)and A36 proteins (lane 2) were stained by India ink. Reference: A36proteins immunoblotted with anti-A36 rabbit antiserum (lane 3).

FIG. 5 represents the analysis of the size of the polypeptide (p362)fused to β-galactosidase expressed by recombinant clone a362 (hereafterdefined). This fusion protein is named βgal-p362.

Lysate proteins of E. coli Y1089 lysogenized either by standard λgt11(tracks C and E) or by the same phage carrying the insert coding forp362 (clone a362) (tracks D and F) were fractionated by 7.5%polyacrylamide gel electrophoresis. Tracks C and D and molecular weightstandards (tracks A and B) were stained with Coomassie brilliant blue,whereas tracks E and F were treated with rabbit anti-A36 antiserum andstained with peroxydase-labeled anti-rabbit antiserum.

FIG. 6 represents the evidence of the belonging of the recombinantpolypeptide p362 to the 34 kD protein of the A36 complex.

The TMA complex from M. paratuberculosis was dissociated and its proteincomponents were fractionated by 10% polyacrylamide gel electrophoresisand transblotted to a nitrocellulose membrane. Fractionated proteinswere either stained with India ink (track b) or incubated with rabbitanti-βgal-p362 antiserum (track c). Track a: molecular weight standards.

FIG. 7A (SEQ ID NO:1) represents the nucleic acid sequence encompassingthe nucleic acid sequence of FIG. 7B and the one of FIG. 7C.

FIG. 7B (SEQ ID NO:2) represents a sequence homologous to the onerepresented on FIG. 7C.

FIG. 7C (SEQ ID NO:3) represents the base sequence of the M.paratuberculosis genomic fragment present in clone a362 and coding forp362.

It should be noted that the two EcoRI sites [GAATTC] present at bothends of the sequence are a result of the cloning strategy and are notnaturally present in the genomic sequence.

FIG. 8 (SEQ ID NO:5) represents the amino acid sequence andcorresponding nucleotide sequence (SEQ ID NO:4) of the recombinantpolypeptide p362.

It should be noted that the first two amino acids, corresponding to theEcoRI sites in the DNA sequence, are not naturally present in the nativeprotein, but are a result of cloning.

FIG. 9a (SEQ ID NO:6 and SEQ ID NO:7) corresponds to the restriction andgenetic map of the pmTNF-MPH plasmid used in Example II for theexpression of p362 of the invention in E. coli.

FIG. 9b (SEQ ID NO:8) corresponds to the pmTNF-MPH nucleic acidsequence.

On this figure, the origin of nucleotide stretches used to constructplasmid pmTNF-MPH is specified hereafter.

Position

1-208: lambda PL containing EcoRI blunt-MboII blunt fragment of pPL(λ)(Pharmacia)

209-436: synthetic DNA fragment

230-232: initiation codon (ATG) of mTNF fusion protein

236-307: sequence encoding AA 2 to 25 of mature mouse TNF

308-384: multiple cloning site containing His₆ encoding sequence atposition 315-332

385-436: HindIII fragment containing E. coli trp terminator

437-943: rrnBT₁T₂ containing HindIII-SspI fragment from pKK223(Pharmacia)

944-3474: DraI-EcoRI blunt fragment of pAT₁₅₃ (Bioexcellence) containingthe tetracycline resistance gene and the origin of replication.

FIG. 10 (SEQ ID NO:9) represents the complete amino acid sequence of therecombinant polypeptide mTNF-H6-p362. The amino acids 1-26 represent themTNF part, the amino acids from 27-46 correspond to the polylinker part(H6) and the remaining amino acids (47-147) represent the M.paratuberculosis 10 kDa polypeptide (p362).

FIG. 11 represents the DNA sequence (SEQ ID NO:10) containing thenucleic acid coding for the protein of 34 kDa hereabove defined and thecorresponding amino acid sequence (SEQ ID NO:11). Nucleotides arenumbered in the right-hand side margin and amino acids are numberedbelow the protein sequence.

It is to be noted that the arrow before amino acid 200 corresponds tothe third amino acid of FIG. 8, since the first two amino acids of FIG.8 are artficial, corresponding to the EcoRI site from cloning.

Table 5 hereafter corresponds to the complete restriction site analysisof pmTNF-MPH.

TABLE 5 RESTRICTION-SITE ANALYSIS Done on DNA sequence PMTNFMPH. Totalnumber of bases is: 3474. Analysis done on the complete sequence. Listof cuts by enzyme. Acc I: 371 2818 Acy I: 788 2264 2921 3035 3056 AflII: 387 Afl III: 1698 Aha III: 224 Alu I: 386 439 1141 1398 1534 17602382 2785 3441 3456 Alw NI: 1289 Apa I: 345 Apa LI: 1384 Asp 718I: 210Asu I: 341 342 547 676 766 1988 2030 2209 2333 2582 267 2945 3297 Ava I:338 2043 Ava II: 547 1988 2030 2333 2582 2670 Bal I: 2026 Bam BI: 3343093 Bbe I: 2267 2924 3038 3059 Bbv I: 1369 1788 1806 1919 1922 28663255 Bbv I*: 1070 1276 1279 2026 2050 2683 Bbv II: 1875 2738 Bgl I: 23062540 Bin I: 17 342 956 1054 1140 3101 Bin I*: 329 955 1052 2366 3088 BspIII: 908 978 2979 Bsp HI: 2414 Bsp HII: 354 Bst NI: 215 528 638 806 15391552 1673 2028 2411 3340 Cau II: 6 339 340 736 769 1321 1986 2212 29363300 Cfr 10I: 374 2185 2539 2699 3058 3067 3308 Cfr I: 2024 2529 29373069 3173 Cla I: 3446 Cvi JI: 192 265 272 343 350 361 386 400 439 444 47660 678 767 828 844 1141 1170 1213 1224 1289 136 1393 1398 1534 16321658 1676 1687 1760 1779 1979 198 2026 2063 2145 2189 2210 2215 23532363 2382 2423 248 2488 2518 2531 2552 2597 2641 2785 2801 2857 2875 2932947 2985 2999 3071 3140 3175 3298 3322 3441 3456 Cvi QI: 211 3306 DdeI: 135 571 661 717 1015 1424 1888 Dpn I: 11 238 336 950 962 1040 10481059 1134 2010 232 2342 2373 2645 3004 3095 3122 Dra II: 1988 2030 2945Dra III: 295 331 Dsa I: 345 2021 2940 Eco 31I: 615 Eco 47III: 1826 26952976 3238 Eco 57I: 216 Eco 57I*: 1156 Eco 78I: 2265 2922 3036 3057 EcoNI: 198 2845 Eco RI: 309 Eco RII: 213 526 636 804 1537 1550 1671 20262409 3338 Eco RV: 3285 Fnu 4HI: 401 417 532 1084 1290 1293 1358 15011656 1774 177 1795 1908 1911 2040 2054 2061 2064 2183 2262 2307 236 24472532 2697 2748 2855 2889 2892 3170 3173 3244 Fnu DII: 542 1074 1655 18371934 2056 2082 2227 2237 2366 243 2493 2498 2525 2654 2769 3125 Fok I:468 852 3370 Fok I*: 816 2423 2468 3322 Gsu I: 2088 Gsu I*: 2642 Hae I:361 828 844 1224 1676 1687 2026 2423 2480 2552 Hae II: 594 1458 18282267 2697 2924 2978 3038 3059 3240 Hae III: 343 361 678 767 828 844 12241658 1676 1687 202 2210 2423 2480 2531 2552 2641 2875 2939 2947 3071 3173298 Hga I: 160 183 796 2088 2238 2829 Hga I*: 1008 1586 2482 2514 3068Hgi AI: 141 1388 2007 2298 2885 3196 Hgi CI: 210 2179 2263 2702 29203034 3055 3349 3392 Hgi JII: 345 2987 3001 Hha I: 542 593 1074 1183 13571457 1524 1794 1827 2017 205 2115 2266 2525 2656 2696 2771 2923 29773037 3058 321 3239 3371 Hin P1I: 540 591 1072 1181 1355 1455 1522 17921825 2015 205 2113 2264 2523 2654 2694 2769 2921 2975 3035 3056 320 32373369 Hind II: 109 372 2819 Hind III: 384 437 3439 Hinf I: 368 1328 17241799 1944 2165 2463 2617 2837 Hpa II: 5 339 355 375 735 769 1130 13201346 1493 198 2186 2212 2450 2540 2700 2776 2936 3059 3068 3083 330 3309Hph I: 96 140 183 716 967 1953 2174 3028 3073 3355 Hph I*: 8 305 311 317Kpn I: 214 Mae I: 365 952 1205 1981 3240 Mae II: 276 330 751 997 19001924 2513 2569 Mae III: 171 257 1162 1278 1341 2320 2587 3255 3343 MboI: 9 236 334 948 960 1038 1046 1057 1132 2008 232 2340 2371 2643 30023093 3120 Mbo II: 209 475 970 1832 1880 2472 2743 Mbo II*: 1041 2997 MmeI*: 1305 1489 3165 3252 Mnl I: 372 1271 1595 2001 2499 2683 Mnl I*: 210291 350 764 1520 1803 2169 2196 2234 2295 259 Mse I: 181 188 223 388 486817 994 3414 3436 Mst I: 2016 2114 3210 Nae I: 2187 2541 2701 3069 NarI: 2264 2921 3035 3056 Nco I: 345 Nhe I: 3239 Nla III: 168 232 349 382565 620 912 982 1702 1881 201 2222 2279 2294 2422 2539 2725 2764 29102983 3121 346 Nla IV: 212 336 343 549 1631 1670 1989 2032 2146 2181 2212265 2583 2704 2922 2946 3036 3057 3095 3141 3351 339 Nru I: 2498 NspBII: 412 1115 1360 2331 Nsp HI: 382 1702 2910 Pfl MI: 295 2105 2154 PleI: 376 1807 Ple I*: 1322 2831 Pma CI: 331 Ppu MI: 1988 2030 Pss I: 19912033 2948 Rsa I: 212 3307 Sal I: 370 2817 Scr PI: 6 215 339 340 528 638736 769 806 1321 153 1552 1673 1986 2028 2212 2411 2936 3300 3340 Sdu I:141 345 1388 2007 2298 2885 2987 3001 3196 Sec I: 5 338 345 1538 20212099 2301 2934 2940 3339 335 Sfa NI: 650 818 2445 2820 3231 3344 SfaNI*: 420 1601 2038 2433 3054 3066 3255 Sma I: 340 Sph I: 382 2910 SsoII: 4 213 337 338 526 636 734 767 804 1319 153 1550 1671 1984 2026 22102409 2934 3298 3338 Stu I: 361 Sty I: 345 2099 Taq I: 254 371 666 16002202 2343 2818 3131 3446 Taq IIB: 1802 Taq IIB*: 2804 Tth111II: 40 1107Tth111II*: 686 1075 1114 Xba I: 364 Xho II: 9 334 948 960 1046 1057 3093Xma I: 338 Xma III: 2529 Xmn I: 467 Total number of cuts is: 743. Listof non cutting selected enzymes. Aat II, Asu II, Avr II, Bbv II*, Bcl I,Bgl II, Bsp MI* Bss HII, Bst EII, Bst XI, Eco 31I*, Esp I, Hpa I, Mlu IMme I, Nde I, Not I, Nsi I, Pst I, Pvu I, Pvu II Rsr II, Sac I, Sac II,Sau I, Sca I, Sci I, Sfi I Sna BI, Spe I, Spl I, Ssp I, Taq IIA, TaqIIA*, Tth 111I Vsp I, Xca I, Xho I Total number of selected enzymeswhich do not cut: 38

EXAMPLE I

Purification of the TMA Complex of M. paratuberculosis (A36),Characterization of the Proteic Part of A36, Identification of the 34kDa Protein and Development of A36 Based Immunoassay

Materials and Methods

Bacteria:

The following mycobacteria were used: M. paratuberculosis strain 2E and316F (from Dr. F. Saxegaard, National Veterinary Institute, Oslo,Norway; Saregaard F. et al., 1985, “Control of paratuberculosis (Johne'sdisease) in goats by vaccination” 116:439-441); M. avium serotype 4(from Dr. F. Portaels, Institute of Tropical Medicine, Antwerpen,Belgium) (Shaefer W. B., 1965, “Serologic identification andclassification of the atypical mycobacteria by their agglutination”, Am.Rev. Resp. Dis. suppl. 92:85-93); M. bovis strain BCG GL2 (from Dr.Weckx, Pasteur Institute, Brussels, Belgium) and M. phlei strain AM76(from Dr. M. Desmecht, National Institute for Veterinary Research,Brussels, Belgium). The 168 strain of B. subtilis was used as controlATCC no 33234.

Preparation of bacterial cytoplasms:

Bacterial suspensions in buffered saline (100 mg wet weight cells/ml0.15 N NaCl 0.02 M K₂HPO₄ pH 7.5 containing 10 mM phenylmethylsulfonylfluoride) were disrupted by sonication (15 min treatment with a 500-Wultrasonic processor, Vibra cell from Sonics and Materials Inc, Danbury,Conn. USA (3 min sonication for B. subtilis). Homogenates werecentrifuged (5000×g, 10 min, 4° C.), and supernatants (i.e.mycobacterial cytoplasms) were stored at −20° C. and used as sources ofantigens.

Purification of TMA complexes:

The supernatant (about 4.5 mg proteins/ml) was submitted to RNAasedigestion (10 μg enzyme/100 g wet weight bacteria, 30 min, 37° C.) andfractionated by gel exclusion chromatography on Sepharose 6B columns(Pharmacia, Uppsala, Sweden) equilibrated with buffered saline, aspreviously detailed (Cocito C. et al., 1986, “Preparation and propertiesof antigen 60 from Mycobacterium bovis BCG” Clin. Exp. Immunol.66:262-272). TMA complexes (thermostable macromolecular antigencomplexes) were found within the excluded fractions (which contained onthe average 0.5 mg soluble proteins/ml). Solutions of TMA (with 1 mMphenylmethylsulfonyl fluoride as conservative) were stored at −20° C.

Purity of TmA complexes was checked by crossed immunoelectrophoresis,according to the reference systems (Closs O. et al., 1980, “The antigensof Mycobacterium bovis, strain BCG, studied by crossedimmunoelectrophoresis: a reference system” Scand. J. Immunol.12:249-263; Gunnarsson E. et al., 1979, “Analysis of antigens inMycobacterium paratuberculosis” Acta Vet. Scand. 20:200-215).

For this purpose agarose gels (1% type 2 agarose from Sigma, St Louis,Mo.) on glass plates (5 by 7 cm) were used, the top gel containing 200μl of rabbit anti-mycobacterial homogenate. Mycobacterial antigen (10 μlof samples containing 0.5 mg TMA/ml) was applied to a corner well andelectrophoretic runs were made as described (1 h, 8 V/cm, 15° C. in 1stdimension; 3 V/cm, 18 h, 15° C. in 2nd dimension). Slants were washed,dried, stained with Coomassie blue and photographed.

Animal sera:

For production of polyclonal antisera, mycobacterial homogenate or TMApreparations (10 μg soluble proteins/0.5 ml buffered saline emulsifiedwith equal volume of incomplete Freund adjuvant) were repeatedlyinjected (6 inoculations at 1-week intervals) into rabbits bysubcutaneous way.

The antibody titer of the sera was evaluated by an immunoenzymometricprocedure (see below).

Here is thus obtained a polyclonal anti-TMA complex antiserum, moreparticularly anti-A36 antiserum, and a polyclonal anti-homogenateantiserum referred to in the Western blotting test.

Four kinds of sera from bovines either healthy or at different stages ofthe Johne's disease were used:

a) healthy controls with no sign of mycobacterial infection and withnegative tests of coproculture and complement fixation; b) asymptomaticnon-excretory stage I of the disease (a case which appeared negative atthe moment of sampling but became positive later);

c) asymptomatic excretory stage II of the disease (positive coproculturewith no clinical signs of disease); and d) symptomatic excretory stageIII of the disease (with positive complement fixation test). These serawere provided by the National Institute of Veterinary Research (Dr. M.Desmecht, Brussels, Belgium) and the Center of Veterinary Medicine (Dr.B. Limbourg, Erpent, Belgium).

Electrophoretic fractionation and Western blotting of TMA proteins:

The protein moiety of TMA complexes was fractionated by electrophoresison 10% polyacrylamide gels, in the presence of Na dodecyl sulfate(SDS-PAGE procedure) (Laemmli U. K., 1970, “Cleavage of structuralproteins during the assembly of the head of bacteriophage T4” Nature227:680-695). Protein samples (25 μg soluble polypeptides in 50 μl 0.125mM Tris-HCl pH 6.8 containing 5% w/v SDS, 20% v/v glycerol, 10% V:Vβ-mercaptoethanol and 0.05% bromophenol blue) were boiled for 5 min andthen applied to vertical gel slabs. Molecular weight protein markers(Sigma Chem. Co., St Louis, Mo.) were: bovine serum albumin (66 kDa),ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa),carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor(20.1 kDa) and α-lactalbumin (14.2 kDa). Electrophoretic runs (4 h, 50V, 20° C.) were made in a vertical unit (LKB, Bromma, Sweden). Proteinbands were visualized by staining with coomassie brilliant blue.Controls of total cytoplasmic proteins were run in parallel with TMAsamples.

Electrophoresed proteins were transferred from polyacrylamide gels tonitrocellulose membranes (BA 85, Macherey-Nagel, Germany) by the use ofa transblot-unit (217 multiphor 2, LKB, Bramma, Sweden).

Transfer buffer contained 20% methanol, 0.039 M glycine and 0.048 M Trisbase pH 8.8, and runs were made at 10 V for 2 h. Transblotted proteinswere identified by reaction with a primary antibody (either polyclonalrabbit antiserum [1/1500] or bovine serum [1/1001]) and then with alabeled secondary antibody.

Transblotted nitrocellulose sheets were first incubated for 30 min withTBS buffer (0.5 M NaCl, 0.023 M Tris-HCl pH 7.5) containing 3% w/vgelatin and then for 3 h with the primary antibodies diluted with TBSTbuffer (TBS containing 0.05% v/v Tween 20) and 1% w/v gelatin. Afterrepeated washings with TBST, sheets were incubated for 2 h withsecondary IgG (1/400 diluted preparations of peroxydase-labeledanti-rabbit, or anti-mouse or anti-cow IgG, Dako, Copenhagen, Denmark),followed by washings with TBST and TBS buffers. A color reaction wasdeveloped by addition of α-chloronaphtol (Bio-Rad Laboratories,Richmond, Calif.) in the presence of hydrogen peroxide. The colorreaction was stopped by washing sheets with distilled water. A similarprotocol was used for antigens directly spotted on nitrocellulosemembranes (dot-blot analysis). Reference samples of transblotted totalproteins and molecular weight markers were visualized by India inkstaining (10% solution of fount India, Pelikan, Germany, in 0.2 M NaCl,0.05 M Tris-HCl pH 7.4 containing 0.3% v/v Tween 20) for 30 min (HancokK. et al., 1983, “India ink staining of proteins on nitrocellulosepaper” Anal. Biochem. 133:157-162).

Immunoassay for determination of anti-mycobacterial Ig:

Multiwell microtiter plates (Microwell Module, Nunc, Denmark) werecoated either with purified A36 or with M. paratuberculosis totalcytoplasm (i.e. supernatant) (0.5 μg soluble proteins/50 μl 0.05 M Nacarbonate buffer pH 9.6/well). Air dry wells were saturated with bovineserum albumin (0.1% w/v BSA in 0.15 M NaCl, 1 h, 37° C.). Increasingdilutions of serum to be tested in 0.15 M NaCl 0.02 M Na phosphatebuffer pH 7.2 0.005% Tween 80 (PBST buffer) were added (50 μl/well, 1 h,37° C.), optimal dilutions being identified by checker board titration.Horse-radish peroxydase-labeled swine anti-rabbit, or rabbit anti-cowantiserum (Dako, Copenhagen, Denmark) were added (50 μl of 1/400 IgGdilution in PBST/well, 1 h, 37° C.). Excess reagent was removed by 5buffer washings. After incubation with the peroxidase reagent (50 μl perwell of a 17 mE Na citrate buffer pH 6.3 containing 0.2% O-phenylenediamine and 0.015% H₂O₂, 30 min, 37° C. in the dark), the reaction wasstopped (50 μl 2 M H₂SO₄) and samples were spectrometrically measured(Plate reader SLT 210 from Kontron Analytical, U.K.). Results wererecorded as ELISA absorbance values (A_(450 nm)).

In some experiments, cross-reactive Ig were removed by incubation (18 h,4° C.) with either purified TMA preparations (0.2 mg protein/ml ofserum) or bacterial homogenates or intact mycobacteria (equivalents of 2mg dry weight bacteria/ml of serum). Absorbed preparations were checkedby dot-blot trials before application in immunoblot or immunoassay.

Immune electron microscopy:

Suspensions of mycobacteria in water (5×10⁷ cells/5 μl) were placed ancarbon-formvar 200-mesh copper grids and air dried. Grids were seriallyincubated with: a) bovine serum albumin (3% solution in buffered saline,30 min, 37° C.); b) anti-TMA complex rabbit antiserum (a 10⁻³ dilutionof Ig in buffered saline with 0.05% Tween 20, 2 h, 37° C.); c) sheepanti-rabbit biotinylated Ig (1/200 dilution of Ig from Amersham, U.K.,in buffered saline-Tween, 1 h, 20° C.);

d) gold-labeled streptavidin (a 1/20 dilution of a preparation fromAmersham, U.K.) (Cloeckaert A. et al., 1990, “Identification of sevensurface-exposed Brucella outer membrane proteins by use of monoclonalantibodies: immunogold labeling for electron microscopy andenzyme-linked immunosorbent assay” Infect. Immun. 58:000-000). Gridswere analyzed in a transmission electron microscope (Philips CM 10).

Results

Purification of TMA complexes and preparation of anti-TMA antisera:

The TMA complex of M. paratuberculosis (A36) has been prepared from thetotal homogenate. Cytoplasm fractionation by gel exclusionchromatography yielded said TMA complex within the exclusion fraction.The immunoelectrophoretic patterns of total cytoplasmic antigens(supernatant) (FIG. 1(1)) and of the exclusion fraction (FIG. 1(2)) arecompared. From these tracings, which were obtained with polyclonalantisera elicited by inoculation of rabbits with whole mycobacterialhomogenate, the purity of the A36 preparation can be inferred.

A similar protocol was used for preparation of other antigens of the TMAgroup from M. avium, M. bovis and M. phlei, which were used forcomparative analysis.

The polyclonal antisera corresponding to the TMA complexes have alsobeen prepared. The purity of these Ig preparations was checked bycrossed immunoelectrophoresis: using total cell homogenates as antigensin every case, a single immunoprecipitogen line corresponding to the TMAcomplex was obtained (patterns not shown, mimicking that of FIG. 1(2)).It is to be noted that subcutaneous injection of TMA complexpreparations invariably induced the synthesis of high titer antisera(ELISA absorbance higher than 2.5 for dilutions at 10⁻⁵), a result whichstressed the high immunogenicity of these antigen complexes.

Development of A36-based serological assay for paratuberculosis:

The availability of A36 has prompted the development of an enzymometricELISA-type immunoassay for paratuberculosis. Accordingly, multiwellplates were coated with A36 and incubated with sera of infected animals.Peroxidase-labeled rabbit anti-bovine IgG were added as second antibody,and the color developed after addition of peroxydase reagent wasmeasured spectrophotometrically, as detailed in Materials and Methods. Acomparative survey was made in parallel with A36 and with totalcytoplasm (supernatant) of M. paratuberculosis (equal amounts ofproteins were used for the two assays).

All the sera of infected animals (stages II and III of the Johne'sdisease) yielded a positive answer (values of 0.84 to 2.25 units) toboth types of the ELISA assay (FIG. 3). On the contrary, uninfectedanimals were invariably negative (values lower than 0.38 units). WithA36-ELISA, considerably higher absorbance values (1.44 to 2.25 units)were obtained than with the total cytoplasm-ELISA (0.84 to 1.65).

These results suggest the immunodominance of the A36 antigen in theJohne's disease, and the usefulness of the A36-based ELISA as adiagnostic assay.

Peripheral location of the TMA complex in mycobacteria:

The observed immunodominance of A36 is more compatible with a surfacecomponent than with an antigen complex located in the cytoplasm.However, a transfer of TMA complex through the envelope and itsprotrusion at the cell surface is conceivable.

The use of the immunoelectron microscopy methodology has allowed adirect approach to this problem. Multiplying cells of M.paratuberculosis were incubated with anti-A36 Ig from immunized rabbits.Cell-bound primary antibodies were revealed by secondary swineanti-rabbit IgG labeled with colloidal gold. Electron micrographs showthe presence of antigen reactive spots on the surface of mycobacteria(results not shown).

These data indicate that part of the TMA complex does indeed occurwithin the cell wall and is presented on the cell surface.

Imunological crosareactivity of A36 and other TMA antigens:

In the preceding section, the development of a A36-based ELISA assay fortitration of anti-mycobacterial antibodies has been described. Thepossible use of this assay in veterinary Medicine relies on itsspecificity with respect to: a) other mycobacteria which are usual hostsof the intestinal tracts of ruminants; and b) M. bovis, and M.tuberculosis which can cause tuberculosis in cattle (compulsoryslaughtering of PPD-positive cattle). This problem was approached byevaluating the crossreactivity of TMA complexes from differentmycobacteria with two procedures (see Table 1).

A first series of assays was carried out with microtitration platescoated with the TMA complex from M. avium, M. bovis, M. paratuberculosisand M. phlei. All these plates were used to titrate a single anti-A36antiserum, a procedure yielding an evaluation of the percentage ofshared TMA epitopes. Considering the autologous reaction (A36-anti A36IgG) equal to 100, percentage of homology of M. paratuberculosis TMAcomplex with the TMA complex of M. avium and bovis was very high; it wasmuch lower for M. phlei TMA complex.

When the A36-based ELISA assay was repeated with anti-A36 antiserumpreviously absorbed by different mycobacterial TMA complexes, anevaluation of the A36 specific epitopes was obtained. From Table 1, itresults that the percentage of specific epitopes was low when the A36was compared to the TMA of M. avium and M. bovis, it was high whencompared to the TMA of M. phlei.

TABLE 1 CROSSREACTING AND SPECIES SPECIFIC EPITOPES IN THE TMA COMPLEXESOF FOUR MYCOBACTERIA TMA in ELISA Coating Absorbing Epitopes (%) reagentreagent ELISA units Cross- Parameter (plate)^(a) (antiserum)^(b)(A_(450 nm))^(c) reacting specific^(d) A. Crossreactivity M. parat. —2.367 100 M. avium — 2.376 (±0.247) 100 (±13) M. bovis — 2.240 (±0.181)96 (±10) M. phlei — 1.083 (±0.156) 49 (±8) B. Specificity M. parat. M.parat. 0.462 0 M. parat. M. avium 0.574 (±0.197) 7 (±11) M. parat. M.bovis 0.603 (±0.238) 10 (±13) M. parat. M. phlei 1.073 (±0.141) 48 (±8)^(a)TMA preparations from different mycobacteria (0.5 μg/well) were usedto coat microtitration plates ^(b)anti-A36 antiserum was pre-absorbed(samples B) or not (samples A) with TMA complex from differentmycobacteria ^(c)to plates coated with A36 (samples B) or with differentTMAs (samples A) anti-A36 antiserum (1/150000 dilution) was added, andbound Ig were revealed by a second labeled antibody ^(d)percentage ofcrossreacting or specific epitopes calculated on a logarithmic scale.

These results show the lack of species-specificity of the A36-ELISA as adiagnostic reagent for the Johne's disease. They suggest, however, thepossible occurrence of A36 components endowed with such a specificity.

Immunodominance and specificity of the A36 proteins:

The species specificity, which was missing at the level of the completeA36 antigen complex, was sought with respect to its proteins components.The TMA complexes from M. avium, M. bovis, M. paratuberculosis and M.phlei were isolated, and their protein components were fractionated bypolyacrylamide electrophoresis. A similarity of M. avium and M.paratuberculosis tracks is apparent, whereas those of M. bovis and M.phlei TMA were clearly different to the M. paratuberculosis track.

When fractionated A36 proteins were immunoblatted with anti-A36antiserum, a dozen of major polypeptides were stained, most of themlocated in the 28-42 kDa region. Immunoblotting with anti-A36 antiserumpre-absorbed with a lysate of M. phlei yielded 5 polypeptide bands; theywere 3 in the case of M. bovis and one with M. avium. Table 2 provides acomparative evaluation of the main A36 components according to twoproperties: immunogenicity level (staining intensity by pooled sera ofinfected bovines) and species specificity (lack of cross-reactivity withthe other mycobacteria). Eleven major components of 22 to 74 kDa arelisted: two of them (of 23 and 31 kDa) containing specific epitopes withrespect to the tested organisms except M. avium, and one of 34 kDacontaining specific epitopes with respect to all of the tested organismsincluding M. avium.

TABLE 2 IMMUNOLOGICAL CHARACTERISTICS OF SOME PROTEINS OF THE TMACOMPLEX OF MYCOBACTERIUM PARATUBERCULOSIS (A36) Immunogenicity^(b,c)Pro- (level in hosts) tein^(a) rabbit infected bovines Specificity^(d)towards (kDa) anti-A36 I II III M. avium M. bovis M. phlei 74 ++ − − +no no no 52 + − − − no no no 41 + + + + no no yes 40 +++ + + + no no no37 ++ ++ − ++ no no yes 35 + ++ ++ ++ no no yes 34 +++ +++ +++ +++ yesyes yes 31 ++ +++ − +++ no yes yes 29 +++ − − + no no yes 23 +++ − + −no yes yes 22 + − ++ − no no yes ^(a)A36 was dissociated and proteincomponents were fractionated by SDS-PAGE electrophoresis and identifiedby immunoblotting ^(b)degree of immunogenicity for rabbits and cows wasevaluated from the intensity of immunoblot staining with thecorresponding sera ^(c)sera from cattle affected by different stages ofthe Johne's disease: I, asymptomatic-non excretory; II,asymptomatic-excretory; and III, symptomatic-excretory forms^(d)crossreactivity was expressed by a no, and specificity by a yes.

The immunological relevance of the latter protein was checked byimmunoblot analysis of A36 proteins with infected bovine sera: a majorband at the level of the 34 kDa marker was observed (FIG. 4, lanes 4, 5,6 and 7). This band was missing in the control (lane 8 with healthybovine serum).

It is thus evident that the 34 kDa protein component of the TMA complexis immunodominant in cattle, relevant to Johne's disease, and containingspecies-specific epitopes with respect to related mycobacteria.

The present invention enables to develop a A36 based ELISA test forparatuberculosis: its ability to reveal the presence of a mycobacterialinfection in cattle has been proven in FIG. 3. Basic requirements forthe use of a given antigen as reagent for immunoassays of medicalinterest are: 1) its immunodominance; 2) its relevance to the targeteddisease; and 3) its specificity. Requirements 1 and 2 were thereforefulfilled by the A36 based-ELISA. Requirements 1 to 3 are completelyfulfilled by the p362 polypeptide which is part of the 34 kDa proteinbelonging to A36, as described hereafter.

EXAMPLE II

Isolation of Clone a362 Expressing a 10 kDa Polypeptide (p362), DNASequencing of the Insert of Clone a362 and Testing of p362 in an ELISAfor Johne's Disease

Material and Methods

Cloning Vectors

The following types were used: λgt11 (Young R. A. and Davis R. W., 1983,“Yeast RNA polymerase II genes: isolation with antibody probes” Proc.Natl. Acad. Sci. USA 80:1195-1198) and pUEX2 (Brennan G. M. et al.,1987, pUEX, a bacterial expression vector related to pEX with universalhost specificity” Nucl. Acids Res. 15:10056) and pmTNF-MPH (see FIGS.9a, 9 b and Table 5) as expression vectors, and the Blue-Script SK⁺assequencing vector (Stratagene).

Bacteria

Mycobacterium paratuberculosis 19698 (from the American Type CultureCollection). M. paratuberculosis: strain 2887 (Crohn): ATCC n^(o) 43015.M. avium serotype 4, M. avium serotype 2, M. avium serotype 8 (SchaeferW. B., 1965, “Serologic identification and classification of theatypical mycobacteria by their agglutination” Am. Rev. Resp. Dis. suppl.92:85-93). M. tuberculosis H37rv: ATCC n^(o) 25618. M. gordonae: ATCCn^(o) 14470. Brucella abortus B3 (Cloeckaent A. et al., 1990, Infect.Immun. 58:3980-3987). Strains of Escherichia coli: Y1089 (Δ(lacU169),Δ(lon), hflA150 (chr::Tn10), (pMC9), (rK⁻, mK⁺)), Y1090 (Δ(lacU169),Δ(lon), sup F, (trpC22::Tn10), (pMC9), (rK⁻, mK⁺)), MC1061 (Δ(lacX74),galU⁻, galK⁻, (rK⁻, mK⁺)) and DH5αF′ (F′, (rK⁻, mK⁺), supE44, lacZΔM15,Δ(lacZYA argF) U169), K12ΔH, ATCC 33767 (lacZ(am) Δ(bio uvr B) (λ Nam7am53 cI 857 ΔH1) rpsL20).

Antisera

Rabbit anti-M. paratuberculosis antiserum was from Dako (Copenhagen,Denmark, lot n^(o) 014). Sera from paratuberculosis-infected cattle wereprovided by Dr. M. Desmecht (National Institute for Veterinary Research,Brussels) and Dr. B. Limbourg (Erpent, Center of Veterinary Medicine,Belgium).

Polyclonal antisera against whole homogenate of M. avium serotype 4, M.bovis BCG, and M. phlei, as well as those against the TMA complex andβgal-p362 (recombinant polypeptide of the invention fused toβ-galactosidase hereafter described) were produced by repeatedsubcutaneous inoculations into rabbits (10 μg proteins/0.5 ml bufferedsaline emulsified with equal volume of incomplete Freund's adjuvant, 6inoculations at 1-week intervals).

Purification of M. paratuberculosis DNA

Suspensions of bacteria (10 mg in 0.5 ml of 100 mM NaCl, 1 mM EDTA, 50mM Tris-HCl pH 7.4) were incubated sequentially with lysozyme (25 μl of20 mg/ml, 14 h, 50° C.), pronase (25 μl of 20 mg/ml, 1 h, 37° C.), andSDS (25 μl of 20%, 1 h 37° C.). Mixtures were extracted withchloroform-isoamyl alcohol (24:1, vol:vol), water-saturated phenol, andether. After incubation with ribonuclease (5 μl of 2 mg/ml, 1 h, 37°C.), DNA was purified on columns of Sephadex G50 (equilibrated with 4.8mM sodium phosphate pH 6.8) and hydroxyapatite (washed with 8 M urea,0.1 M sodium phosphate buffer pH 6.8 containing 1% SDS, and then with4.8 M sodium phosphate pH 6.8, and eluted with 480 mM sodium phosphatepH 6.8).

Construction of a λgt11 Library of M. paratuberculosis

M. paratuberculosis DNA was sheared to average length segments of 0.5 to1.5 kb (Vibra Cell ultrasonicator 60 W, 2 sec). Shearing was monitoredby agarose gel electrophoresis. EcoR1 sites were methylated with EcoR1methylase (5 μg of sheared DNA in 50 μl of buffer (50 mM Tris-HCl pH7.5, 1 mM Na₃EDTA, 5 mM dithiothreitol, 50 μM S-adenosyl-L-methionineand 10 units of EcoR1 methylase). Methylation was pursued for 30 min at37° C., and stopped by 10 min incubation at 70° C. Blunt-end DNAfragments were obtained by incubation with T4 DNA polymerase (5 μl of0.1 M MgCl₂, 2.5 μl of 1 mM dTNPs, 1 μl of 1 M(NH₄)₂SO₄, and 20 units ofT4 DNA polymerase per 40 μl methylation reaction medium; 20 minincubation at 37° C.). EDTA (15 mM final concentration) was added,reaction mixture was extracted with phenol/chloroform twice, and theaqueous. phase was extracted with ether. After addition of sodiumacetate 0.3 M final concentration, DNA was precipitated with 2 vol ofEtOH at −20° C. and washed with 70% EtOH. DNA pellet was dissolved inbuffer (10 μl of 100 mM Tris-HCl pH 7,5, 20 mM MgCl₂, 20 mMdithiothreitol), phosphorylated EcoR1 linkers (200 μg/ml) were added,followed by addition of PEG 6000 (final concentration 15%), 1 mM ATP(final concentration) and 2 units of T4 DNA ligase, and the reactionmixture was incubated overnight at 12° C. This mixture was incubated at37° C. with an excess of EcoR1, and DNA fragments were purified fromlinker excess on Sephadex G25. The DNA solution thus obtained wasextracted sequentially with phenol/chloroform and ether, precipitated,and washed with ethanol. DNA pellet (0.5 μg) was dissolved in TE buffer(10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and ligated (18 h, 4° C.) with 1 μgof dephosphorylated EcoR1-digested λgt11 DNA (Promega). Methylation,ligation, and digestion steps were controlled by agarose gelelectrophoresis. Phage packaging of cloned DNA was obtained with theStratagene gigapack extract.

Screening of the λgt11 Library and Dot-blot Technique

After infection of E. coli Y1090 by the recombinant phage mixture andspreading them out over the plate, they were incubated for 3-4 h at 42°C.

For identification of recombinant phages, IPTG (isopropylthioβ-galactopyranoside) (10 mM) saturated nitrocellulose filters wereplaced directly on the surface of the overlay plates containing theplaques and incubated for 18 h at 37° C. (Young R. A. and Davis R. W.,1983, “Yeast RNA polymerase II: genes: isolation with antibody probes”Proc. Natl. Acad. Sci. USA 80:1195-1198). After spotting of controlantigens (1 μg) and washing for 10 min with TBS buffer (0.5 M NaCl,0.023 M Tris-HCl pH 7.5), filters were incubated for 30 min with thesame buffer containing 3% (w/v) gelatin and then with the rabbit anti-M.paratuberculosis antiserum (Dako) previously diluted with TBST buffer(TBS buffer containing 0,05% (v/v) Tween 20) containing 1% (w/v)gelatin. After washing, filters were incubated for 1 h with 1/400dilutions of peroxydase-labeled anti-rabbit Ig. After repeated washingwith TBST and TBS, the peroxydase substrate α-chloronaphtol (Bio RadLaboratories, Richmond, Calif.) and hydrogen peroxide were added.Reaction was stopped by washing with distilled water. Plaquescorresponding to reactive spots on the filters were picked off,transferred to SM medium (100 mM NaCl, 10 mM MgSO₄, 20 mM Tris-HCl pH7.4) and purified by repeated passages in E. coli Y1090. Recombinantclones were then further characterized with respect to theirantigenicity (incubation with bovine sera and anti-A36) and theirspecificity (incubation with antibodies directed against homogenate ofM. avium, M. bovis and M. phlei) using the same procedure as describedabove.

A similar technique was used for dot-blot experiments in which thespecificity of the recombinant polypeptide p362 was tested with respectto different mycobacteria: spots of mycobacterial homogenates onnitrocellulose membranes were incubated with anti-βgal-p362 Ig.

High Level Expression of Fusion Protein in E. coli

Colonies of E. coli Y1089 lysogenized with the appropriate λgt11recombinants were multiplied at 30° C. in Luria-Bertani medium(A_(6000 nM)=0.5). After heat shock (20 min at 45° C.), production ofβ-galactosidase fusion proteins of the invention was induced by theaddition of 10 mM IPTG (final concentration) and further incubation (60min at 37° C.). Cells harvested by centrifugation were suspended inbuffer (10 mM Tris-HCl, pH 8.2, 2 mM EDTA) and rapidly frozen in liquidnitrogen.

For enhanced expression, λgt11 inserts were subcloned into theexpression vector pUEX2 (Brennan G. M. et al., 1987, “pUEX, a bacterialexpression vector related to pEX with universal host specificity” Nucl.Acids Res. 15:10056), commercially available from Amersham, which was.used to transform E. coli MC1061 (Maniatis, Molecular Cloning). Singlecolonies of transformed E. coli were grown at 30° C. to A₆₀₀=0.3 andheat-shocked (90 min at 42° C.). Harvested cells were lysed bysonication and frozen in liquid nitrogen.

Protein Fractionation and Immunoblotting

The TMA complex and recombinant proteins were analyzed by polyacrylamidegel electrophoresis under denaturing conditions (SDS PAGE) (Laemmli, U.K. 1970, “Cleavage of structural proteins during the assembly of thehead of bacteriophage T4”, Nature 227:680-695).

Fractionation on 7.5 or 10% acrylamide gels was carried out in a 2001vertical electrophoresis unit (LKB-Produkter AB, Bromma, Sweden) (4 h,50 V, 20° C.). Molecular weight protein markers (Sigma, St Louis, Mo.)were: myosin (205 kDa), β-galactosidase (116 kDa), phosphorylase B (97,4kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa),glyceraldehyde-3-phosphate dehydrogenase (36 kDa) carbonic anhydrase (29kDa), trypsinogen (24 kDa), trypsin inhibitor (20.1 kDa), andα-lactalbumin (14.2 kDa). Protein bands were stained with Coomassiebrilliant blue. Electrophoresed proteins were transblotted (LKB 217Multiphor 2 Electrophoresis System, 10 V, 2 h, with buffer 20% methanol,0.039 M glycine and 0,048 M Tris base, pH 8.8) onto nitrocellulosemembranes. Mycobacterial antigens were visualized by sequentialincubation with polyclonal rabbit antisera (anti-A36 for recombinantmycobacterial antigens fused to β-galactosidase or anti-βgal-p362 forTMA proteins) and peroxydase-labeled anti-rabbit Ig (Dako, Copenhagen,Denmark) (1/400 dilution). Total protein blotted on the membrane wasvisualized by staining with India ink.

DNA Sequencing

Sequence analysis of the DNA insert of the recombinant clone a362 wasdone by the primer extension and dideoxy termination method (Sanger F.et al., 1977, “DNA sequencing with chain terminating inhibitors”, Proc.Natl. Acad. Sci. USA 74:5463-5467), after subcloning of the λgt11 insertinto the sequencing vector pBluescript SK⁺ (Stratagene). Sequencingreactions were performed with T7 DNA polymerase and different primers(universal, reverse, SK, and KS primers from Deaza Kit, Pharmacia,Uppsala, Sweden). Computer-aided analysis of nucleic acid andpolypeptide sequences were performed with the program COD-FICK (PC-GENE,Intelligenetics, USA). Homology searches were performed on DNA level inEMBL bank (release 29) and UGEN bank (release 70-29)(IntelligeneticsInc., CA-USA), and on protein level in PIR bank (release 31) and SwissProt (release 20). No homologous sequences were found.

Serological Analysis (ELISA) with Recombinant Polypeptides

Multiwell microtiter plates (Microwell Module, High binding Capacity,Nunc, Denmark) were coated with total cytoplasm of E. coli-a362 and withtotal cytoplasm of E. coli as a control. Four μg of soluble proteins/50μl 0.05 M Na carbonate buffer pH 9.6 were coated per well. Plates wereair dried overnight and saturated (0.1% serum albumin in 0.15 M NaCl, 1h at 37° C.). Dilutions of bovine Ig in PBST (0.15 M NaCl, 0.02 Mphosphate buffer pH 7.2, containing 0.005% Tween 80) were added to platewells (50 μl, 1 h at 37° C.), Peroxydase-labelled rabbit anti-cow Ig(Dako) (50 μl, 1/400 dilution in PBST/per well) were added (1 h at 37°C.). Excess of reagent was removed by 5 PBST washings. After incubationwith peroxydase reagent (50 μl/well of 0.2% O-phenylenediamine with0.015% hydrogen peroxyde in 0.017 M Na citrate buffer pH 6.3, 30 min,37° C. in the dark), the reaction was stopped with 50 μl 2 M H₂SO₄, andA_(450 nm) was measured in a calorimetric plate reader (SLT 210, KontronAnalytical, UK). Results were recordered as ELISA absorbance values. Insome experiments, cross reactive Ig were removed by incubation (18 h at4° C.) with bacterial homogenate. Absorbed preparations were checked bydot-blot trials before applications in immunoblots or immunoassays.

Immune Electron Microscopy

Suspensions of mycobacteria in water (5×10⁷ cells/5 μl) were placed oncarbon-formvar 200-mesh copper grids and air-dried. Grids were seriallyincubated with: a) bovine serum albumin (3% solution in buffered saline,30 min, 37° C.); b) anti-βgal-p362 rabbit antiserum (a 10⁻³ dilution ofIg in buffered saline with 0.05% Tween 20, 2 h, 37° C.); c) sheepanti-rabbit biotinylated Ig (1/200 dilution of Ig from Amersham, U.K.,in buffered saline-Tween, 1 h, 20° C.); d) gold-labelled streptavidin (a1/20 dilution of a preparation from Amersham, U.K.) (Cloeckaert A. etal., 1990, “Identification of seven surface-exposed Brucella outermembrane proteins by use of monoclonal antibodies: immunogold labelingfor electron microscopy and enzyme-linked immunosorbent assay”, Infec.Immun. 58:3980-3987).

Grids were analyzed in a transmission electron microscope (Philips CM10).

Results

1. Preparation of a Genomic Library of M. paratuberculosis and Isolationof Recombinant Clones

A genomic library of M. paratuberculosis has been prepared by the use ofthe expression vector λgt11. For this purpose, purified mycobacterialDNA was sonicated under controlled conditions yielding segments of 10³ Lon the average (0.5 to 2×10³). These fragments were methylated by EcoR1DNA methylase (efficiency of methylation was controlled by incubationwith EcoR1), incubated with T4 DNA polymerase to obtain blunt-end DNA,and provided with EcoR1 linkers by incubation with T4 DNA ligase. AfterEcoR1 digestion, DNA segments were purified free of linker excess andinserted into EcoR1-cleaved λgt11 by incubation with T4 DNA ligase (astep checked by gel electrophoresis). After packaging and infection ofE. coli Y1090, 7.5×10⁵ recombinant clones (75% of total clones) wereobtained, one third of which was screened with rabbit anti-M.paratuberculosis antiserum (Dako). After repeated purifications, tenrecombinant clones were selected: three of them expressed TMA complexproteins, and seven produced epitopes of proteins not present within theTMA complex.

2. Analysis of Antigenicity and Specificity of Polypeptides Produced byRecombinant Clones

Since cloning of M. paratuberculosis genes was aimed at producingpolypeptides to be used as diagnostic reagents, it appeared essential totest the reactivity of recombinant clones towards sera of cattleaffected by the Johne's disease. As shown in Table 3, all the selectedclones reacted with sera of animals bearing one of the clinical forms ofthe disease. The strongest reactions were afforded by clones a4 anda362. On the contrary, no reactivity was observed with sera from healthybovines.

TABLE 3 Characteristics of clones expressing an antigenic polypeptide ofM. paratuberculosis Antigenicity** Specificity with respect to** Clones*1 2 3 M. avium M. bovis M. phlei a1  (+) + + no no yes a2  + + + yes yesyes a3  + + ++ no yes yes a4  ++ ++ ++ no no yes a5  + + + no yes yesa6  + + ++ no no yes a7  (+) + + no no no a361 + + ++ no yes yes a362 ++++ ++ yes yes yes a363 (+) + + no no yes *only clones a361 to a363express polypeptides belonging to the TMA complex. **detected by serafrom asymptomatic and non excretory bovine (1), asymptomatic andexcretory bovine (2) and symptomatic and excretory bovine (3);quantified as low reaction “(+)”, good reaction “+” and very goodreaction “++”. ***cross reactivity was expressed by a “no”, andspecificity by a “yes”.

Another requirement of paramount importance was the specificity withrespect to mycobacteria belonging to the saprophytic and pathogenicflora of cattle. Recombinant clones were tested for reactivity withantisera against homogenates of M. avium, M. bovis and M. phlei. It waspreviously shown that the overall DNA homology levels of these threemycobacteria with respect to M. paratuberculosis were respectively 94,52, and 19 percent (Hurley S. S. et al., 1988, “DNA relatedness of M.paratuberculosis to other members of the family of mycobacteriaceae”,Int. Journal Syst. Bact. 38:143-146). Data in Table 3 indicate that,although all clones but one were specific towards M. phlei, only five ofthem were specific for M. bovis and two for M. avium.

In conclusion, only two of the selected clones, a2 and a362 fulfilledboth requirements for species-specificity and relevance to Johne'sdisease. Moreover, only the latter clone reacted with anti-A36 antiserumand corresponded, therefore, to a A36 protein, presumably the 34 kDaprotein previously identified as a TMA complex component withspecies-specific epitopes. The remaining part of this example relates tothe characterization and use of clone a362.

3. Size of Clone a362 Insert and its Expressed Polypeptide P362

EcoR1 cleavage of DNA of clone a362 yielded an insert of about 500 bpdevoid of internal EcoR1 restriction sites (not shown).

E. coli Y1089 was lysogenized by the recombinant phage, and thesynthesis of a chimaeric protein fused with β-galactosidase was inducedby IPTG: a fusion protein of about 125 kDa (βgal-p362) was produced(FIG. 5). Since β-galactosidase (116 kDa) misses 2 kDa in λgt11, therecombinant polypeptide coded for by the insert of clone a362 (p362) isexpected to be about 11 kDa in size. Consequently, only a roughly 300 bpportion of the 500 bp insert coded for such an 11 kDa polypeptide. Thiswas confirmed by sequencing and determination of the orientation of theinsert DNA as described further.

4. Production of P362 Recombinant Polypeptide and Evidence of itsBelonging to a 34 kDa Protein of A436

Since the production of the β-gal p362 by E. coli Y1089 containing theλgt11-recombinant phage was only 2% of total proteins, the correspondinginsert was recloned in a more favorable expression vector. For thispurpose, the λgt11 insert of the a362 recombinant clone was freed byincubation with EcoR1, purified by electroelution from an agarose gel(75% recovery), and recloned into the EcoR1 site of the expressionvector pUEX2 (Amersham). In this case, production of βgal-p362 fusionprotein in the transformed MC1061 strain of E. coli (6×10⁵transformants/μg DNA) was about 25% of total proteins.

After running the SDS-PAGE of the lysate from the transformed strains,the recombinant fusion protein was eluted from the polyacrylamide geland used to elicit antibodies in rabbits (anti-βgal-p362).

The protein components of the TMA complex from M. paratuberculosis werefractionated by electrophoresis on polyacrylamide gels (SDS PAGE). Aftertransfer to nitrocellulose sheets, TMA proteins were incubated withanti-βgal-p362. As shown in FIG. 6, a major band corresponding to the 34kDa protein of the TMA complex was immunolabeled: this was the uniqueTMA protein containing species-specific epitopes as above reported. Asecond band of about 31 kDa was stained to minor extent: it was alsopresent in the immunoblots of TMA proteins with sera of infected cattle.

5. Localization of the P362 Polypeptide at the Bacterial Surface

Since the A36 antigen complex was previously shown to be present at thecell surface, a peripheral location of the p362 recombinant polypeptidewould further confirm the belonging of p362 recombinant polypeptide to aprotein of the A36 complex. Electron micrographs show indeed thepresence of the p362 polypeptide within the cell wall and its releaseduring the declining growth phase (results not shown).

6. Assessment of the Species-specificity of the Recombinant Polypeptidep362

From what is above-mentioned, it is shown that the 34 kDa proteincomponent of the TMA complex of M. paratuberculosis contains epitopesdevoid of crossreactivity towards M. bovis, M. avium and M. phlei.Although the recombinant p362 polypeptide, which apparently represents aportion of the 34 kDa protein, is likely to be endowed ofspecies-specificity, a more stringent confirmation is needed for apolypeptide forecast as reagent for serological tests. Consequently, thespecificity of p362 was tested against two series of M. paratuberculosisand M. avium isolates from cattle as well as against certainGram-positive and Gram-negative bacteria being usual hosts of bovine gut(Table 4).

The dot-blot experiment was carried out by spotting on a nitrocellulosemembrane 2 μg samples of different bacterial homogenates. Membranes werethen incubated successively with rabbit anti-βgal-p362 antiserum and,after washing, with peroxydase-labeled swine anti-rabbit IgG. Spots wererevealed by the peroxydase reaction. All of eight M. paratuberculosisisolates were positive, whereas the closely related organisms of theMAIS group were negative. None of the other tested mycobacteria gave apositive reaction, neither did the Nocardia and Brucella species (seeTable 4).

TABLE 4 SPECIFICITY OF p362 TOWARDS OTHER [MYCO]BACTERIA Bacteriumlysates Anti-βgal-p362 Bacterium lysates Anti-βgal-p362 M.paratuberculosis: 2E + M. intracellulare (1) − 316F + MAIS A3 (4) − ATCC19698 + MAIS A84 (4) − ATCC 43015 + MAIS 8715 (4) − 2890 (bovine) (1) +MAIS 87537 (4) − 2891 (bovine) (1) + M. bovis BCG GL2 − 2895 (goat)(1) + M. tuberculosis H37rv (6) − 172 28/66 (bovine) (2) + M. phlei AM76(1) − M. avium D4(5) − M. leprae (1) − M. avium serotype 4 − M.fortuitum M62 (1) − M. avium serotype 8 − M. smegmatis (1) − M. aviumserotype 2 − M. gordonae ATCC 14430 − M. scrofulaceum (1) − Nocardiaasteroides (1) − Salmonella typhimurium (3) − Brucella abortus B3(3) −(+) positive immunological reaction (−) absence of reaction (1) PortaelsIMTA (Institut de Médecine Tropicale, Anvers Belgique) (2) fromKaeckenbeeck DBUL (Département de Bactériologie, Université de Liège,Belgique) (3) from LIMET ICP (Institut of Cellular Pathology, Belgique)(4) from Defoe IPB (Institut Pasteur du Brabant, Belgique) (5) fromSaxegaard NVIN (National Veterinary Institute, Norway). (6) ATCC 25618

7. Sequencing of the Cloned Insert Coding for Polypeptide p362

To sequence the 500 bp DNA segment coding for the polypeptide p362, theinsert of clone a362 was isolated by EcoR1 cleavage from the chimaericvector λgt11 and recloned into the Bluescript vector SK⁺. Aftertransformation of E. coli DH5αF′, clones carrying inserts coding forp362 were selected.

The sequence of the insert showed the occurrence of a 507 bp DNA segmentflanked by two EcoR1 extremities (FIG. 7C (SEQ ID NO:3)). The G+Ccontent of this segment was 70%, in agreement with the 64% G+C of thewhole M. paratuberculosis genome. The sequence in FIG. 7C (SEQ ID NO:3)yielded two open reading frames in phase with the EcoRI sites: a 306 bpregion (1 to 306) in one direction, and a 185 bp region (507 to 322)into opposite orientation. The program COD-FICK (PC-GENE) which takes inaccount the codon usage, confirmed the coding ability of the two openreading frames. They coded respectively for 10 kDa and 7 kDapolypeptides. The insert was subcloned in an expression vector in E.coli in both orientations. only one orientation yielded an expressionproduct reacting with the rabbit anti-βgal-p362 antiserum. Restrictionanalysis led to the selection of the 306 bp open reading frame as beingthe one coding for the p362 polypeptide [10 kDa]. The selected codingregion and the aninoacid sequence of polypeptide p362, corresponding tothe carboxyterminal extremity of the 34 kDa protein are displayed inFIG. 8.

8. Testing of p362 in an ELISA for Johne's Disease

The 10 kDa polypeptide (p362), endowed with species-specificity, andbeing part of the 34 kDa protein of A36, can be used as a specific testfor paratuberculosis.

A preliminary test has been done using plates coated with totalcytoplasm of E. coli-a362 containing p362. Bovine sera were preabsorbedto E. coli-control homogenate. FIG. 2 shows that all sera from infectedbovines react significantly with p362. On the contrary, healthy bovines(samples 26-32) do not give a signal which is significantly higher thanthat observed with E. coli-control cytoplasm.

Antibodies directed against p362 are already present in the early stagesof the disease (samples 1-13). p362 can thus be considered as a verysuitable antigen for specific and sensitive diagnosis ofparatuberculosis.

To decrease the background levels due to cross reaction with theβ-galactosidase part of the fusion protein, the insert coding for p362was recloned into another expression vector (pmTNF-MPH, Innogenetics)(FIGS. 9a and 9 b)(SEQ ID NOS:6,7 and 8).

It contains the tetracycline resistance gene and the origin ofreplication of pAT₁₅₃ (obtainable from Bioexcellence, Biores B. V.,Woerden. The Netherlands), the lambda PL promoter up to the MboII sitein the N gene 5′ untranslated region (originating from pPL(λ);Pharmacia), followed by a synthetic ribosome binding site (see sequencedata), and the information encoding the first 25 AA of mTNF (except forthe initial Leu which is converted to Val). This sequence is, in turn,followed by a synthetic polylinker sequence which encodes sixconsecutive histidines followed by several proteolytic sites (a formicacid, CNBr, kallikrein, and E. coli protease VII sensitive site,respectively), each accessible via a different restriction enzyme whichis unique for the plasmid (SmaI NcoI, BspMII and StuI, respectively; seerestriction and genetic map, FIG. 9a) (SEQ ID NO:6 and 7). Downstreamfrom the polylinker, several transcription terminators are presentincluding the E. coli trp terminator (synthetic) and the rrnBT₁T₂(originating from pKK223-3; Pharmacia). The total nucleic acid sequenceof this plasmid is represented in FIG. 9b (SEQ ID NO:8).

Table 5 gives a complete restriction site analysis of pmTNF-MPH.

The presence of 6 successive histidines allows purification of thefusion protein by Immobilized Metal Ion Affinity Chromatography (IMAC).

To subclone the insert coding for p362 in pmTNF-MPH, it was set freefrom the construct in vector pUEX2 by EcoRI digestion. The EcoRIfragment (507 bp) was eluted from the gel, purified, blunted andinserted in the blunted XbaI site of pmTNF-MPH. The resultingrecombinant plasmid, pmTNF-MPH-a362, is brought into E. coli strainK12ΔH (ATCC 33767) by transformation. After growth at 28° C., expressionof the recombinant protein is induced by a temperature shift to 42° C.,which is held on during 2 hours. Cells were harvested, centrifuged andlysed in French press.

The expressed fusion protein mTNF-H6-p362, present in the cytoplasmfraction of the E. coli recombinant, is purified by Immobilized MetalIon Affinity Chromatography (IMAC) using conditions known by the manskilled in the art. The amino acid sequence of this complete fusionprotein is represented in FIG. 10 (SEQ ID NO:9).

The purified fusion protein is used to coat 96-well microtitrationplates, which were incubated with serial dilutions of sera fromuninfected (control) and infected animals. Plate bound IgG were titratedwith peroxydase-labeled rabbit anti-bovine IgG, as described inMaterials and Methods.

18 508 base pairs nucleic acid double linear DNA (genomic) Mycobacteriumparatuberculosis unsure 35 unsure 185 unsure 252 1 GAATTCCCGG GTGGTCAGCAGCATTCGCCG CAGGNCTACG GGTCGCAGTA CGGCGGTTAC 60 GGCCAGGGCG GCGCTCCGACCGGCGGTTTC GGTGCCCAGC CGTCGCCGCA GTCCGGCCCG 120 CAACAGTCCG CGCAGCAGCAGGGCCCGTCC ACACCGCCCA CCGGCTTCCC CAGCTTCAGC 180 CCGCNGCCCA ACGTCGGCGGGGGATCGGAC TCCGGTTCGG GGACCGCCAA TTACTCCGAG 240 CAGGCCGGTG GNCCAGCAGTCCTACGGCCA GGAGCCTTCT TCACCGTCTG GGCCGACGCC 300 CGCCTAACGT GCCCTGTCGCGCCTAGTCGG GAACGTGCCC CAGAGTGACA CGGGTGGAGG 360 ACAACCGGGC AGCGGGCGCTCGCCAGGCGC GTGACCTCGT CAGGGTCGCG TTCGCCCCGG 420 CGGTGGTGGC ACTGGTCATCATCGCCGCGG TCACGCTGAT CCAGTTGTTG ATCGCCAACA 480 GCGACATGAC CGGCGCGTTGGGGAATTC 508 507 base pairs nucleic acid double linear DNA (genomic)Mycobacterium paratuberculosis 2 GAATTCCCGG GTGGTCAGCA GCATTCGCCGCAGGCTACGG GTCGCAGTAC GGCGGTTACG 60 GCCAGGGCGG CGCTCCGACC GGCGGTTTCGGTGCCCAGCC GTCGCCGCAG TCCGGCCCGC 120 AACAGTCCGC GCAGCAGCAG GGCCCGTCCACACCGCCCAC CGGCTTCCCC AGCTTCAGCC 180 CGCGGCCCAA CGTCGGCGGG GGATCGGACTCCGGTTCGGC GACCGCCAAT TACTCCGAGC 240 AGGCCGGTGG CCCAGCAGTC CTACGGCCAGGAGCCTTCTT CACCGTCTGG GCCGACGCCC 300 GCCTAACGTG CCCTGTCGCG CCTAGTCGGGAACGTGCCCC AGAGTGACAC GGGTGGAGGA 360 CAACCGGGCA GCGGGCGCTC GCCAGGCGCGTGACCTCGTC AGGGTCGCGT TCGCCCCGGC 420 GGTGGTGGCA CTGGTCATCA TCGCCGCGGTCACGCTGATC CAGTTGTTGA TCGCCAACAG 480 CGACATGACC GGCGCGTTGG GGAATTC 507507 base pairs nucleic acid double linear DNA (genomic) Mycobacteriumparatuberculosis 3 GAATTCCCGG GTGGTCAGCA GCATTCGCCG CAGGGCTACGGGTCGCAGTA CGGCGGTTAC 60 GGCCAGGGCG GCGCTCCGAC CGGCGGTTTC GGTGCCCAGCCGTCGCCGCA GTCCGGCCCG 120 CAACAGTCCG CGCAGCAGCA GGGCCCGTCC ACACCGCCCACCGGCTTCCC CAGCTTCAGC 180 CCGCCGCCCA ACGTCGGCGG GGGATCGGAC TCCGGTTCGGCGACCGCCAA TTACTCCGAG 240 CAGGCCGGTG GCCAGCAGTC CTACGGCCAG GAGCCTTCTTCACCGTCTGG GCCGACGCCC 300 GCCTAACGTG CCCTGTCGCG CCTAGTCGGG AACGTGCCCCAGAGTGACAC GGGTGGAGGA 360 CAACCGGGCA GCGGGCGCTC GCCAGGCGCG TGACCTCGTCAGGGTCGCGT TCGCCCCGGC 420 GGTGGTGGCA CTGGTCATCA TCGCCGCGGT CACGCTGATCCAGTTGTTGA TCGCCAACAG 480 CGACATGACC GGCGCGTTGG GGAATTC 507 306 basepairs nucleic acid single linear DNA (genomic) Mycobacteriumparatuberculosis CDS 1..306 mat_peptide 1..303 4 GAA TTC CCG GGT GGT CAGCAG CAT TCG CCG CAG GGC TAC GGG TCG CAG 48 Glu Phe Pro Gly Gly Gln GlnHis Ser Pro Gln Gly Tyr Gly Ser Gln 1 5 10 15 TAC GGC GGT TAC GGC CAGGGC GGC GCT CCG ACC GGC GGT TTC GGT GCC 96 Tyr Gly Gly Tyr Gly Gln GlyGly Ala Pro Thr Gly Gly Phe Gly Ala 20 25 30 CAG CCG TCG CCG CAG TCC GGCCCG CAA CAG TCC GCG CAG CAG CAG GGC 144 Gln Pro Ser Pro Gln Ser Gly ProGln Gln Ser Ala Gln Gln Gln Gly 35 40 45 CCG TCC AGA CCG CCC ACC GGC TTCCCC AGC TTC AGC CCG CCG CCC AAC 192 Pro Ser Arg Pro Pro Thr Gly Phe ProSer Phe Ser Pro Pro Pro Asn 50 55 60 GTC GGC GGG GGA TCG GAC TCC GGT TCGGCG ACC GCC AAT TAC TCC GAG 240 Val Gly Gly Gly Ser Asp Ser Gly Ser AlaThr Ala Asn Tyr Ser Glu 65 70 75 80 CAG GCC GGT GGC CAG CAG TCC TAC GGCCAG GAG CCT TCT TCA CCG TCT 288 Gln Ala Gly Gly Gln Gln Ser Tyr Gly GlnGlu Pro Ser Ser Pro Ser 85 90 95 GGG CCG ACG CCC GCC TAA 306 Gly Pro ThrPro Ala 100 101 amino acids amino acid linear protein 5 Glu Phe Pro GlyGly Gln Gln His Ser Pro Gln Gly Tyr Gly Ser Gln 1 5 10 15 Tyr Gly GlyTyr Gly Gln Gly Gly Ala Pro Thr Gly Gly Phe Gly Ala 20 25 30 Gln Pro SerPro Gln Ser Gly Pro Gln Gln Ser Ala Gln Gln Gln Gly 35 40 45 Pro Ser ArgPro Pro Thr Gly Phe Pro Ser Phe Ser Pro Pro Pro Asn 50 55 60 Val Gly GlyGly Ser Asp Ser Gly Ser Ala Thr Ala Asn Tyr Ser Glu 65 70 75 80 Gln AlaGly Gly Gln Gln Ser Tyr Gly Gln Glu Pro Ser Ser Pro Ser 85 90 95 Gly ProThr Pro Ala 100 60 base pairs nucleic acid single linear DNA (genomic)Mycobacterium paratuberculosis CDS 1..60 mat_peptide 1..57 6 CAG GGA ATTCAC CAT CAC CAT CAC CAC GTG GAT CCC GGG CCC ATG GCT 48 Gln Gly Ile HisHis His His His His Val Asp Pro Gly Pro Met Ala 1 5 10 15 TTC CGG AGGCCT 60 Phe Arg Arg Pro 20 20 amino acids amino acid linear protein 7 GlnGly Ile His His His His His His Val Asp Pro Gly Pro Met Ala 1 5 10 15Phe Arg Arg Pro 20 3474 base pairs nucleic acid double linear DNA(genomic) 8 AATTCCGGGG ATCTCTCACC TACCAAACAA TGCCCCCCTG CAAAAAATAAATTCATATAA 60 AAAACATACA GATAACCATC TGCGGTGATA AATTATCTCT GGCGGTGTTGACATAAATAC 120 CACTGGCGGT GATACTGAGC ACATCAGCAG GACGCACTGA CCACCATGAAGGTGACGCTC 180 TTAAAAATTA AGCCCTGAAG AAGGGCAGGG GTACCAGGAG GTTTAAATCATGGTAAGATC 240 AAGTAGTCAA AATTCGAGTG ACAAGCCTGT AGCCCACGTC GTAGCAAACCACCAAGTGGA 300 GGAGCAGGGA ATTCACCATC ACCATCACCA CGTGGATCCC GGGCCCATGGCTTTCCGGAG 360 GCCTCTAGAG TCGACCGGCA TGCAAGCTTA AGTAAGTAAG CCGCCAGTTCCGCTGGCGGC 420 ATTTTNNTTG ATGCCCAAGC TTGGCTGTTT TGGCGGATGA GAGAAGATTTTCAGCCTGAT 480 ACAGATTAAA TCAGAACGCA GAAGCGGTCT GATAAAACAG AATTTGCCTGGCGGCAGTAG 540 CGCGGTGGTC CCACCTGACC CCATGCCGAA CTCAGAAGTG AAACGCCGTAGCGCCGATGG 600 TAGTGTGGGG TCTCCCCATG CGAGAGTAGG GAACTGCCAG GCATCAAATAAAACGAAAGG 660 CTCAGTCGAA AGACTGGGCC TTTCGTTTTA TCTGTTGTTT GTCGGTGAACGCTCTCCTGA 720 GTAGGACAAA TCCGCCGGGA GCGGATTTGA ACGTTGCGAA GCAACGGCCCGGAGGGTGGC 780 GGGCAGGACG CCCGCCATAA ACTGCCAGGC ATCAAATTAA GCAGAAGGCCATCCTGACGG 840 ATGGCCTTTT TGCGTTTCTA CAAACTCTTT TGTTTATTTT TCTAAATACATTCAAATATG 900 TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT AATAAAAGGATCTAGGTGAA 960 GTCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTTCCACTGAGCG 1020 TTCAGACCCC GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTCTGCGCGTAAT 1080 CTGCTGCTTG CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGCCGGATCAAGA 1140 GCTACCAACT CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATACCAAATACTGT 1200 CCTTCTAGTG TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCACCGCCTACATA 1260 CCTCGCTCTG CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGTCGTGTCTTAC 1320 CGGGTTGGAC TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCTGAACGGGGGG 1380 TTCGTGCACA CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGATACCTACAGCG 1440 TGAGCATTGA GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGTATCCGGTAAG 1500 CGGCAGGGTC GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACGCCTGGTATCT 1560 TTATAGTCCT GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGTGATGCTCGTC 1620 AGGGGGGCGG AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGTTCCTGGCCTT 1680 TTGCTGGCCT TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTGTGGATAACCG 1740 TATTACCGCC TTTGAGTGAG CTGATACCGC TCGCCGCAGC CGAACGACCGAGCGCAGCGA 1800 GTCAGTGAGC GAGGAAGCGG AAGAGCGCTG ACTTCCGCGT TTCCAGACTTTACGAAACAC 1860 GGAAACCGAA GACCATTCAT GTTGTTGCTC AGGTCGCAGA CGTTTTGCAGCAGCAGTCGC 1920 TTCACGTTCG CTCGCGTATC GGTGATTCAT TCTGCTAACC AGTAAGGCAACCCCGCCAGC 1980 CTAGCCGGGT CCTCAACGAC AGGAGCACGA TCATGCGCAC CCGTGGCCAGGACCCAACGC 2040 TGCCCGAGAT GCGCCGCGTG CGGCTGCTGG AGATGGCGGA CGCGATGGATATGTTCTGCC 2100 AAGGGTTGGT TTGCGCATTC ACAGTTCTCC GCAAGAATTG ATTGGCTCCAATTCTTGGAG 2160 TGGTGAATCC GTTAGCGAGG TGCCGCCGGC TTCCATTCAG GTCGAGGTGGCCCGGCTCCA 2220 TGCACCGCGA CGCAACGCGG GGAGGCAGAC AAGGTATAGG GCGGCGCCTACAATCCATGC 2280 CAACCCGTTC CATGTGCTCG CCGAGGCGGC ATAAATCGCC GTGACGATCAGCGGTCCAGT 2340 GATCGAAGTT AGGCTGGTAA GAGCCGCGAG CGATCCTTGA AGCTGTCCCTGATGGTCGTC 2400 ATCTACCTGC CTGGACAGCA TGGCCTGCAA CGCGGGCATC CCGATGCCGCCGGAAGCGAG 2460 AAGAATCATA ATGGGGAAGG CCATCCAGCC TCGCGTCGCG AACGCCAGCAAGACGTAGCC 2520 CAGCGCGTCG GCCGCCATGC CGGCGATAAT GGCCTGCTTC TCGCCGAAACGTTTGGTGGC 2580 GGGACCAGTG ACGAAGGCTT GAGCGAGGGC GTGCAAGATT CCGAATACCGCAAGCGACAG 2640 GCCGATCATC GTCGCGCTCC AGCGAAAGCG GTCCTCGCCG AAAATGACCCAGAGCGCTGC 2700 CGGCACCTGT CCTACGAGTT GCATGATAAA GAAGACAGTC ATAAGTGCGGCGACGATAGT 2760 CATGCCCCGC GCCCACCGGA AGGAGCTGAC TGGGTTGAAG GCTCTCAAGGGCATCGGTCG 2820 ACGCTCTCCC TTATGCGACT CCTGCATTAG GAAGCAGCCC AGTAGTAGGTTGAGGCCGTT 2880 GAGCACCGCC GCCGCAAGGA ATGGTGCATG CAAGGAGATG GCGCCCAACAGTCCCCCGGC 2940 CACGGGGCCT GCCACCATAC CCACGCCGAA ACAAGCGCTC ATGAGCCCGAAGTGGCGAGC 3000 CCGATCTTCC CCATCGGTGA TGTCGGCGAT ATAGGCGCCA GCAACCGCACCTGTGGCGCC 3060 GGTGATGCCG GCCACGATGC GTCCGGCGTA GAGGATCCAC AGGACGGGTGTGGTCGCCAT 3120 GATCGCGTAG TCGATAGTGG CTCCAAGTAG CGAAGCGAGC AGGACTGGGCGGCGGCCAAA 3180 GCGGTCGGAC AGTGCTCCGA GAACGGGTGC GCATAGAAAT TGCATCAACGCATATAGCGC 3240 TAGCAGCACG CCATAGTGAC TGGCGATGCT GTCGGAATGG ACGATATCCCGCAAGAGGCC 3300 CGGCAGTACC GGCATAACCA AGCCTATGCC TACAGCATCC AGGGTGACGGTGCCGAGGAT 3360 GACGATGAGC GCATTGTTAG ATTTCATACA CGGTGCCTGA CTGCGTTAGCAATTTAACTG 3420 TGATAAACTA CCGCATTAAA GCTTATCGAT GATAAGCTGT CAAACATGAGAATT 3474 147 amino acids amino acid linear peptide 9 Met Val Arg SerSer Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His 1 5 10 15 Val Val AlaAsn His Gln Val Glu Glu Gln Gly Ile His His His His 20 25 30 His His ValAsp Pro Gly Pro Met Ala Phe Arg Arg Pro Leu Glu Phe 35 40 45 Pro Gly GlyGln Gln His Ser Pro Gln Gly Tyr Gly Ser Gln Tyr Gly 50 55 60 Gly Tyr GlyGln Gly Gly Ala Pro Thr Gly Gly Phe Gly Ala Gln Pro 65 70 75 80 Ser ProGln Ser Gly Pro Gln Gln Ser Ala Gln Gln Gln Gly Pro Ser 85 90 95 Thr ProPro Thr Gly Phe Pro Ser Phe Ser Pro Pro Pro Asn Val Gly 100 105 110 GlyGly Ser Asp Ser Gly Ser Ala Thr Ala Asn Tyr Ser Glu Gln Ala 115 120 125Gly Gly Gln Gln Ser Tyr Gly Gln Glu Pro Ser Ser Pro Ser Gly Pro 130 135140 Thr Pro Ala 145 1839 base pairs nucleic acid single linear DNA(genomic) Mycobacterium paratuberculosis CDS 742..1638 mat_peptide742..1635 10 GGGCCCGAAC TTGACGAACT CGCCGTCGTA GCTGGCTTCC TCGTCGGTCCACAGCGCCCG 60 CATCGCTTCC AGGTATTCGC GCAGCATGGT GCGGCGCCGG CCCGCCGGCACGCCGTGGTC 120 GGCGAGTTCG TCGGTGTTCC AGCCGAACCC GACGCCGAGG CTGACCCGGCCGCCGGACAG 180 ATGGTCAAGG GTGGCAATAC TTTTCGCCAG CGTGATCGGG TCGTGTTCGACCGGCAGGGC 240 CAGCGCGGTG GACAGCCGCA CCCGCGAGGT GACGGCACAG GCCGCGCCCAGACTGACCCA 300 CGGGTCCAGG GTGCGCATGT AGCGGTCGTC GGGCAGCGAC GCGTCGCCGGTGGTCGGGTG 360 CGCGGCCTCC CGCTTGATCG GGATATGCGT GTGTTCCGGC ACGTAGAAGGTCGCAAACCC 420 GTGGTCGTCG GCAAGCTTCG CGGCCGCAGC CGGAGAGATG CCACGGTCGCTGGTGAAAAG 480 CACAAGCCCG TAATCCATGC AGTGAATTAG AACGTGTTCT ACCTCTGCGGGGCAAGCTGT 540 CGTGATACGG ACCGTCTCGC CGCGCGGTCG TCTGCGAAGC CCGCGGGCAAGCCAATGGCG 600 ACGGCACCGG CCGTCGCACG TGCGCTAGCG TGGGTGATCG ACCGTGTCGCTCGCGCAGTG 660 ACGCGCCTGC AAGCACCGCG TCGCATCGCA ACCGTGGCGC CCGCTCGGCACTAAAAGGCA 720 GTGGAAGCAA CAGGAGGAGC C ATG ACC TAC TCT CCC GGC AGC CCCGGA TAT 771 Met Thr Tyr Ser Pro Gly Ser Pro Gly Tyr 1 5 10 CCA CCG GCGCAG TCT GGC GGC ACC TAT GCA GGC GCC ACA CCA TCT TTC 819 Pro Pro Ala GlnSer Gly Gly Thr Tyr Ala Gly Ala Thr Pro Ser Phe 15 20 25 GCC AAA GAC GACGAC GGC AAG AGC AAA CTC CCG CTC TAC CTC AAC ATC 867 Ala Lys Asp Asp AspGly Lys Ser Lys Leu Pro Leu Tyr Leu Asn Ile 30 35 40 GCC GTG GTC GCC CTGGGT TTC GCG GCC TAC CTG CTG AAT TTC GGC CCC 915 Ala Val Val Ala Leu GlyPhe Ala Ala Tyr Leu Leu Asn Phe Gly Pro 45 50 55 ACC TTC ACC ATC GGC GCCGAC CTC GGC CCG GGT ATC GGC GGC CGC GCG 963 Thr Phe Thr Ile Gly Ala AspLeu Gly Pro Gly Ile Gly Gly Arg Ala 60 65 70 GGT GAC GCC GGC ACC GCC GTCGTG GTG GCG CTG CTG GCC GCG CTG CTC 1011 Gly Asp Ala Gly Thr Ala Val ValVal Ala Leu Leu Ala Ala Leu Leu 75 80 85 90 GCC GGG CTG GGC CTG CTG CCCAAG GCC AAG AGT TAT GTG GGC GTG GTC 1059 Ala Gly Leu Gly Leu Leu Pro LysAla Lys Ser Tyr Val Gly Val Val 95 100 105 GCG GTC GTC GCG GTC CTC GCCGCG CTG CTG GCC ATC ACC GAG ACG ATC 1107 Ala Val Val Ala Val Leu Ala AlaLeu Leu Ala Ile Thr Glu Thr Ile 110 115 120 AAC CTG CCC GCC GGT TTC GCGATC GGC TGG GCG ATG TGG CCG CTG GTG 1155 Asn Leu Pro Ala Gly Phe Ala IleGly Trp Ala Met Trp Pro Leu Val 125 130 135 GCG TGC GTG GTG CTG CAG GCGATC GCC GCG GTG GTC GTG GTC CTG CTG 1203 Ala Cys Val Val Leu Gln Ala IleAla Ala Val Val Val Val Leu Leu 140 145 150 GAC GCC GGG GTG ATC ACG GCGCCG GCG CCG CGG CCC AAG TAC GAC CCC 1251 Asp Ala Gly Val Ile Thr Ala ProAla Pro Arg Pro Lys Tyr Asp Pro 155 160 165 170 TAC GCG CAG TAC GGC CAATAC GGG CAA TAC GGC CAG TAC GGG CAA CAG 1299 Tyr Ala Gln Tyr Gly Gln TyrGly Gln Tyr Gly Gln Tyr Gly Gln Gln 175 180 185 CCC TAC TAC GGT CAG CCGGGC GGT CAG CCC GGG GGC CAG CCG GGT GGT 1347 Pro Tyr Tyr Gly Gln Pro GlyGly Gln Pro Gly Gly Gln Pro Gly Gly 190 195 200 CAG CAG CAT TCG CCG CAGGGC TAC GGG TCG CAG TAC GGC GGT TAC GGC 1395 Gln Gln His Ser Pro Gln GlyTyr Gly Ser Gln Tyr Gly Gly Tyr Gly 205 210 215 CAG GGC GGC GCT CCG ACCGGC GGT TTC GGT GCC CAG CCG TCG CCG CAG 1443 Gln Gly Gly Ala Pro Thr GlyGly Phe Gly Ala Gln Pro Ser Pro Gln 220 225 230 TCC GGC CCG CAA CAG TCCGCG CAG CAG CAG GGC CCG TCC ACA CCG CCC 1491 Ser Gly Pro Gln Gln Ser AlaGln Gln Gln Gly Pro Ser Thr Pro Pro 235 240 245 250 ACC GGC TTC CCC AGCTTC AGC CCG CCG CCC AAC GTC GGC GGG GGA TCG 1539 Thr Gly Phe Pro Ser PheSer Pro Pro Pro Asn Val Gly Gly Gly Ser 255 260 265 GAC TCC GGT TCG GCGACC GCC AAT TAC TCC GAG CAG GCC GGT GGC CAG 1587 Asp Ser Gly Ser Ala ThrAla Asn Tyr Ser Glu Gln Ala Gly Gly Gln 270 275 280 CAG TCC TAC GGC CAGGAG CCT TCT TCA CCG TCT GGG CCG ACG CCC GCC 1635 Gln Ser Tyr Gly Gln GluPro Ser Ser Pro Ser Gly Pro Thr Pro Ala 285 290 295 TAACGTGCCCTGTCGCGCCT AGTCGGGAAC GTGCCCCAGA GTGACACGGG TGGAGGACAA 1695 CCGGGCAGCGGGCGCTCGCC AGGCGCGTGA CCTCGTCAGG GTCGCGTTCG CCCCGGCGGT 1755 GGTGGCACTGGTCATCATCG CCGCGGTCAC GCTGATCCAG TTGTTGATCG CCAACAGCGA 1815 CATGACCGGCGCGTTGGGGA ATTC 1839 298 amino acids amino acid linear protein 11 MetThr Tyr Ser Pro Gly Ser Pro Gly Tyr Pro Pro Ala Gln Ser Gly 1 5 10 15Gly Thr Tyr Ala Gly Ala Thr Pro Ser Phe Ala Lys Asp Asp Asp Gly 20 25 30Lys Ser Lys Leu Pro Leu Tyr Leu Asn Ile Ala Val Val Ala Leu Gly 35 40 45Phe Ala Ala Tyr Leu Leu Asn Phe Gly Pro Thr Phe Thr Ile Gly Ala 50 55 60Asp Leu Gly Pro Gly Ile Gly Gly Arg Ala Gly Asp Ala Gly Thr Ala 65 70 7580 Val Val Val Ala Leu Leu Ala Ala Leu Leu Ala Gly Leu Gly Leu Leu 85 9095 Pro Lys Ala Lys Ser Tyr Val Gly Val Val Ala Val Val Ala Val Leu 100105 110 Ala Ala Leu Leu Ala Ile Thr Glu Thr Ile Asn Leu Pro Ala Gly Phe115 120 125 Ala Ile Gly Trp Ala Met Trp Pro Leu Val Ala Cys Val Val LeuGln 130 135 140 Ala Ile Ala Ala Val Val Val Val Leu Leu Asp Ala Gly ValIle Thr 145 150 155 160 Ala Pro Ala Pro Arg Pro Lys Tyr Asp Pro Tyr AlaGln Tyr Gly Gln 165 170 175 Tyr Gly Gln Tyr Gly Gln Tyr Gly Gln Gln ProTyr Tyr Gly Gln Pro 180 185 190 Gly Gly Gln Pro Gly Gly Gln Pro Gly GlyGln Gln His Ser Pro Gln 195 200 205 Gly Tyr Gly Ser Gln Tyr Gly Gly TyrGly Gln Gly Gly Ala Pro Thr 210 215 220 Gly Gly Phe Gly Ala Gln Pro SerPro Gln Ser Gly Pro Gln Gln Ser 225 230 235 240 Ala Gln Gln Gln Gly ProSer Thr Pro Pro Thr Gly Phe Pro Ser Phe 245 250 255 Ser Pro Pro Pro AsnVal Gly Gly Gly Ser Asp Ser Gly Ser Ala Thr 260 265 270 Ala Asn Tyr SerGlu Gln Ala Gly Gly Gln Gln Ser Tyr Gly Gln Glu 275 280 285 Pro Ser SerPro Ser Gly Pro Thr Pro Ala 290 295 11 amino acids amino acid linearpeptide 12 Glu Phe Pro Gly Gly Gln Gln His Ser Pro Gln 1 5 10 17 aminoacids amino acid linear peptide 13 Gln Gln Ser Tyr Gly Gln Glu Pro SerSer Pro Ser Gly Pro Thr Pro 1 5 10 15 Ala 307 base pairs nucleic aciddouble linear DNA (genomic) 14 GAATTCCCGG GTGGTCAGCA GCATTCGCCGCAGGNCTACG GGTCGCAGTA CGGCGGTTAC 60 GGCCAGGGCG GCGCTCCGAC CGGCGGTTTCGGTGCCCAGC CGTCGCCGCA GTCCGGCCCG 120 CAACAGTCCG CGCAGCAGCA GGGCCCGTCCACACCGCCCA CCGGCTTCCC CAGCTTCAGC 180 CCGCNGCCCA ACGTCGGCGG GGGATCGGACTCCGGTTCGG GGACCGCCAA TTACTCCGAG 240 CAGGCCGGTG GNCCAGCAGT CCTACGGCCAGGAGCCTTCT TCACCGTCTG GGCCGACGCC 300 CGCCTAA 307 306 base pairs nucleicacid double linear DNA (genomic) 15 GAATTCCCGG GTGGTCAGCA GCATTCGCCGCAGGCTACGG GTCGCAGTAC GGCGGTTACG 60 GCCAGGGCGG CGCTCCGACC GGCGGTTTCGGTGCCCAGCC GTCGCCGCAG TCCGGCCCGC 120 AACAGTCCGC GCAGCAGCAG GGCCCGTCCACACCGCCCAC CGGCTTCCCC AGCTTCAGCC 180 CGCGGCCCAA CGTCGGCGGG GGATCGGACTCCGGTTCGGC GACCGCCAAT TACTCCGAGC 240 AGGCCGGTGG CCCAGCAGTC CTACGGCCAGGAGCCTTCTT CACCGTCTGG GCCGACGCCC 300 GCCTAA 306 306 base pairs nucleicacid double linear DNA (genomic) 16 GAATTCCCGG GTGGTCAGCA GCATTCGCCGCAGGGCTACG GGTCGCAGTA CGGCGGTTAC 60 GGCCAGGGCG GCGCTCCGAC CGGCGGTTTCGGTGCCCAGC CGTCGCCGCA GTCCGGCCCG 120 CAACAGTCCG CGCAGCAGCA GGGCCCGTCCACACCGCCCA CCGGCTTCCC CAGCTTCAGC 180 CCGCCGCCCA ACGTCGGCGG GGGATCGGACTCCGGTTCGG CGACCGCCAA TTACTCCGAG 240 CAGGCCGGTG GCCAGCAGTC CTACGGCCAGGAGCCTTCTT CACCGTCTGG GCCGACGCCC 300 GCCTAA 306 597 base pairs nucleicacid single linear DNA (genomic) CDS 1..597 17 ATG ACC TAC TCT CCC GGCAGC CCC GGA TAT CCA CCG GCG CAG TCT GGC 48 Met Thr Tyr Ser Pro Gly SerPro Gly Tyr Pro Pro Ala Gln Ser Gly 1 5 10 15 GGC ACC TAT GCA GGC GCCACA CCA TCT TTC GCC AAA GAC GAC GAC GGC 96 Gly Thr Tyr Ala Gly Ala ThrPro Ser Phe Ala Lys Asp Asp Asp Gly 20 25 30 AAG AGC AAA CTC CCG CTC TACCTC AAC ATC GCC GTG GTC GCC CTG GGT 144 Lys Ser Lys Leu Pro Leu Tyr LeuAsn Ile Ala Val Val Ala Leu Gly 35 40 45 TTC GCG GCC TAC CTG CTG AAT TTCGGC CCC ACC TTC ACC ATC GGC GCC 192 Phe Ala Ala Tyr Leu Leu Asn Phe GlyPro Thr Phe Thr Ile Gly Ala 50 55 60 GAC CTC GGC CCG GGT ATC GGC GGC CGCGCG GGT GAC GCC GGC ACC GCC 240 Asp Leu Gly Pro Gly Ile Gly Gly Arg AlaGly Asp Ala Gly Thr Ala 65 70 75 80 GTC GTG GTG GCG CTG CTG GCC GCG CTGCTC GCC GGG CTG GGC CTG CTG 288 Val Val Val Ala Leu Leu Ala Ala Leu LeuAla Gly Leu Gly Leu Leu 85 90 95 CCC AAG GCC AAG AGT TAT GTG GGC GTG GTCGCG GTC GTC GCG GTC CTC 336 Pro Lys Ala Lys Ser Tyr Val Gly Val Val AlaVal Val Ala Val Leu 100 105 110 GCC GCG CTG CTG GCC ATC ACC GAG ACG ATCAAC CTG CCC GCC GGT TTC 384 Ala Ala Leu Leu Ala Ile Thr Glu Thr Ile AsnLeu Pro Ala Gly Phe 115 120 125 GCG ATC GGC TGG GCG ATG TGG CCG CTG GTGGCG TGC GTG GTG CTG CAG 432 Ala Ile Gly Trp Ala Met Trp Pro Leu Val AlaCys Val Val Leu Gln 130 135 140 GCG ATC GCC GCG GTG GTC GTG GTC CTG CTGGAC GCC GGG GTG ATC ACG 480 Ala Ile Ala Ala Val Val Val Val Leu Leu AspAla Gly Val Ile Thr 145 150 155 160 GCG CCG GCG CCG CGG CCC AAG TAC GACCCC TAC GCG CAG TAC GGC CAA 528 Ala Pro Ala Pro Arg Pro Lys Tyr Asp ProTyr Ala Gln Tyr Gly Gln 165 170 175 TAC GGG CAA TAC GGC CAG TAC GGG CAACAG CCC TAC TAC GGT CAG CCG 576 Tyr Gly Gln Tyr Gly Gln Tyr Gly Gln GlnPro Tyr Tyr Gly Gln Pro 180 185 190 GGC GGT CAG CCC GGG GGC CAG 597 GlyGly Gln Pro Gly Gly Gln 195 199 amino acids amino acid linear protein 18Met Thr Tyr Ser Pro Gly Ser Pro Gly Tyr Pro Pro Ala Gln Ser Gly 1 5 1015 Gly Thr Tyr Ala Gly Ala Thr Pro Ser Phe Ala Lys Asp Asp Asp Gly 20 2530 Lys Ser Lys Leu Pro Leu Tyr Leu Asn Ile Ala Val Val Ala Leu Gly 35 4045 Phe Ala Ala Tyr Leu Leu Asn Phe Gly Pro Thr Phe Thr Ile Gly Ala 50 5560 Asp Leu Gly Pro Gly Ile Gly Gly Arg Ala Gly Asp Ala Gly Thr Ala 65 7075 80 Val Val Val Ala Leu Leu Ala Ala Leu Leu Ala Gly Leu Gly Leu Leu 8590 95 Pro Lys Ala Lys Ser Tyr Val Gly Val Val Ala Val Val Ala Val Leu100 105 110 Ala Ala Leu Leu Ala Ile Thr Glu Thr Ile Asn Leu Pro Ala GlyPhe 115 120 125 Ala Ile Gly Trp Ala Met Trp Pro Leu Val Ala Cys Val ValLeu Gln 130 135 140 Ala Ile Ala Ala Val Val Val Val Leu Leu Asp Ala GlyVal Ile Thr 145 150 155 160 Ala Pro Ala Pro Arg Pro Lys Tyr Asp Pro TyrAla Gln Tyr Gly Gln 165 170 175 Tyr Gly Gln Tyr Gly Gln Tyr Gly Gln GlnPro Tyr Tyr Gly Gln Pro 180 185 190 Gly Gly Gln Pro Gly Gly Gln 195

What is claimed is:
 1. A biologically pure polypeptide consisting of theamino acid sequence of SEQ ID NO 5, or a fragment thereof, wherein saidfragment: is recognized by antibodies also recognizing the sequence ofSEQ ID NO 5, but the fragment is not recognized by antibodies raisedagainst M. bovis, M. avium, M. phlei or M. tuberculosis; as an immunogengives rise to antibodies which recognize the sequence of SEQ ID NO 5 butwhich do not recognize M. bovis, M. avium, M. phlei or M. tuberculosis;and reacts with the majority of sera from cattle suffering from Johne'sdisease.
 2. An isolated polypeptide consisting of any of the followingsequences: the amino acid sequence of 101 amino acids of SEQ ID NO5,Glu-Phe-Pro-Gly-Gly-Gln-Gln-His-Ser-Pro-Gln(SEQ ID NO 12), orGln-Gln-Ser-Tyr-Gly-Gln-Glu-Pro-Ser-Ser-Pro-Ser-Gly-Pro-Thr-Pro-Ala (SEQID NO 13).
 3. A fusion protein comparing an amino acid sequence of thebiologically pure polypeptide according to claim 1 and a heterologousamino acid sequence comprising from 1 to 1,100 amino acids.
 4. A fusionprotein consisting of the amino acid sequence represented in (SEQ ID NO9).
 5. An expression product expressed by cellular host transformed by arecombinant vector comprising regulating elements enabling theexpression of a nucleic acid sequence encoding the polypeptide accordingto claim
 1. 6. The expression product of claim 5, wherein the cellularhost is a bacteria or a eukaryotic microorganism.
 7. A method for the invirto diagnosis of paratuberculosis in an animal or of infection of ananimal by Mycobacterium paratuberculosis comparing the steps of:contacting a biological sample taken from said animal with a fusionprotein according to claim 3, under conditions enabling an in vitroimmunological reaction between said polypeptide and one or moreantibodies recognizing said polypeptide, and decting in vitro theantigen/antibody complex which is formed, when paratuberculosis orinfection by Mycobacterium paratuberculosis is present.
 8. A method forin vitro diagnosis of Crohn's disease in a patient or infection of apatient by Mycobacterium paratuberculosis comprising the steps of:contacting a bilogical sample taken from said patient with a fusionprotein according to claim 3, under conditions enabling an in vitroimmunological reaction between said polypeptide and one or moreantibodies recognizing said polypeptide, and detecting in vitro theantigen/antibdy complex which has been formed, when Crohn's diseases orinfection by Mycobaterium paratuberculosis is present.
 9. An immunogeniccomposition comparing a fusion protein according to claim 3, and apharmaceutically acceptable vehicle.
 10. A method for in vitro diagnosisof paratuberculosis in an animal or of infection of an animal byMycobacterium paratuberculosis comprising the steps of: contacting abiological sample taken from said animal with a biologically purepolypeptide according to claim 1 under condition enabling an in vitroimmunological reaction between said polypeptide and one or moreantibodies recognizing said polypeptide, and decting in vitro theantigen/antibody complex which is formed, when paratuberculosis orinfection by Mycobacterium paratuberculosis is present.
 11. A method forin vitro diagnosis of Crohn's disease in a patient or infection of apatient by Mycobacterium paratuberculosis comprising the steps of:contacting a biological sample taken from said patient with abiologically pure polypeptide according to claim 1 under conditionsenabling an in vitro immunological reaction between said polypeptide andone or more antibodies recognizing said polpeptide, and detecting invitro the antigen/antibody complex which has been formed, when Crohn'sdisease or infection by Mycobacterium paratuberculosis is present. 12.An immunogenic composition comprising a biologically pure polypeptideaccording to claim 1 and a pharmaceutically vehicle.